Hyperbranched polyglycerol-coated particles and methods of making and using thereof

ABSTRACT

Core-shell particles and methods of making and using thereof are described herein. The core is formed of or contains one or more hydrophobic materials or more hydrophobic materials. The shell is formed of or contains hyperbranched polyglycerol (HPG). The HPG coating can be modified to adjust the properties of the particles. Unmodified HPG coatings impart stealth properties to the particles which resist non-specific protein absorption and increase circulation in the blood. The hydroxyl groups on the HPG coating can be chemically modified to form functional groups that react with functional groups and adhere the particles to tissue, cells, or extracellular materials, such as proteins.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a 371 application of International Application No.PCT/US2015/030187, filed May 11, 2016, which claims benefit of U.S.Provisional Application No. 61/991,025, filed May 9, 2014, which isincorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under Grant Nos.EB000487 and CA149128 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

FIELD OF THE INVENTION

This invention is in the field of particles, such as microparticlesand/or nanoparticles, coated with hyperbranched polyglycerol, whereinthe coating can be tuned to provide stealth or adhesive properties.

BACKGROUND OF THE INVENTION

Over the past decade, nanotechnology has been explored to improvebioavailability, lower side effects, and enhance targeting oftherapeutic agents for a wide variety of diseases. When agents areadministered systemically, the therapeutic effect is typically loweredby rapid clearance through enzymatic digestion, renal filtration, andmononuclear phagocytic system (MPS) uptake. Encapsulating the agent innanoparticles (NPs) has been investigated to modulate these factors, asthe precisely engineered NPs can protect the agent from rapid clearancebut also help it reach the target site more efficiently andpreferentially. Widely used materials for producing NPs includepolymers, lipids and some inorganic materials. However, encapsulation oftherapeutic agents in NPs does not ensure successful delivery. In fact,particulates are often more efficiently cleared from the blood by MPSuptake, particularly by phagocytic cells in the liver, leading to rapidloss of NPs and their associated drugs from circulation, which limitstheir ability to reach non-liver targets.

It is well-known that surface modification of NPs with substances thatprevent non-specific adsorption can reduce their interaction with serumproteins and increase the blood circulation of the NPs. An ideal surfacecoating resists non-specific adsorption of proteins and facilitates theattachment of other functionalities, such as targeting ligands, to theparticle. To resist non-specific adsorption in physiological conditions,materials for coating are usually charge neutral, hydrophilic, andstable in physiological environments. Among the few materials used ascoating for NPs, PEG has become ubiquitous. The advantages of PEG as acoating of NPs for drug delivery include its low toxicity, lowimmunogenicity, and resistance to non-specific adsorption ofbiomolecules. PEG has so dominated the field of surface coatings thatnew approaches are rarely investigated.

However, PEG has considerable limitations. For instance, it is knownthat PEG chains can adopt a variety of configurations on the surface,depending on PEG surface density, and the most effective densities areoften difficult to achieve.

There exists a need for particles with improved coatings, in which thecoatings can be tuned to provide stealth or adhesive properties and canfurther be modified with targeting moieties, and which overcome thelimitations associated with polyethylene glycol coatings.

Therefore, it is an object of the invention to provide particles withimproved coatings, in which the coatings can be tuned to provide stealthor adhesive properties and can further be modified with targetingmoieties, and which overcome the limitations associated withpolyethylene glycol coatings.

SUMMARY OF THE INVENTION

Core-shell particles, such as microparticles and nanoparticles, andmethods of making and using, are described herein. The core is formed ofor contains a hydrophobic material or more hydrophobic material, such asa polymer. The shell is formed of or contains hyperbranched polyglycerol(HPG). The HPG can be covalently bound to the one or more materials thatform the core such that upon self-assembly, particles are formed inwhich the hydrophobic or more hydrophobic materials form the core andthe HPG forms a coating on the particle.

The HPG coating can be modified to adjust the properties of theparticles. For example, unmodified HPG coatings impart stealthproperties to the particles which resist non-specific protein absorptionand increase circulation in the blood. Alternatively, the hydroxylgroups on the HPG coating can be chemically modified to form functionalgroups that react with functional groups on tissue or otherwise interactwith tissue to adhere the particles to the tissue, cells, orextracellular materials, such as proteins. Such functional groupsinclude, but not limited to, aldehydes, amines, and O-substitutedoximes.

The surface of the particles can further be modified with one or moretargeting moieties or covalently bound to HPG via a coupling agent orspacer in organic solvents such as dichloromethane (DCM),dimethylformamide (DMF), dimethyl sulfoxide (DMSO) or tetrahydrofuran(THF). In some embodiments, the polymer is functionalized/modifiedbefore nanoparticle formation. Alternatively, the targeting moieties maybe attached to NPs after the synthesis of NPs in aqueous solution orother protic solution such as alcohol. For example, HPG coated NPs canbe transformed to aldehyde terminated NPs by NaIO₄ treatment (orcarboxylic acid terminated by NaIO₄ treatment followed by sodiumchlorite treatment) so the targeting moieties may be directly covalentlyattached to NPs via aldehyde (or carboxylic acid) groups on NPs andfunctional groups (amine, hydrazine, aminooxy and their derivatives) onthe targeting moieties or indirectly attached to the NPs via couplingagents or spacers (such as aminooxy modified biotin and cysteine).

The particles can further contain one or more therapeutic agents,diagnostic agents, prophylactic agents, and/or nutraceuticals. The oneor more agents can be covalently or non-covalently associated with theparticles. The agents can be encapsulated within the particle, forexample, dispersed within the core; non-covalently associated with thesurface of the particles, covalently-associated with the surface of theparticles, or combinations thereof.

The particles are useful in methods for delivery of therapeutic,nutraceutical, diagnostic and prophylactic agents and/or nutraceuticals.

HPG coatings can also be used to alter the surface properties of othermoieties, such as delivery vehicles (liposomes, micelles, proteinaggregates), metals and metal oxides, (thiolated gold conjugated toHPG). HPG can impart stealth properties to these materials.Alternatively, the vicinyl diol groups can be transformed to functionalgroups that promote adhesion of the vehicle to biological materials,such as tissue, cells, and/or proteins.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic showing the synthesis of stealth nanoparticles andsticky nanoparticles.

FIGS. 2A, 2B, 2C and 2D are graphs showing frequency (%) as a functionof particle size for PLA-HPG nanoparticles.

FIG. 3 is a graph showing drug release (%) as a function of time (hours)for PLA-HPG/camptothecin (CPT) and PLA-PEG/CPT nanoparticles.

FIG. 4A is a graph showing cell viability (%) as a function ofcamptothecin (CPT) concentration (μM) for free CPT, PLA-HPG/CPTnanoparticles, and PLA-PEG/CPT nanoparticles. FIG. 4B is a graph showingcell viability (%) as a function of nanoparticle (NP) concentration(mg/ml) for PLA-HPG nanoparticles and PLA-PEG nanoparticles

FIG. 5 is a graph showing dye retention (%) of PLA-HPG and PLA-PEGnanoparticles as a function of incubation time in PBS.

FIG. 6A is a graph showing the dose in blood (%) as a function of time(hours) for PLA-HPG and PLA-PEG nanoparticles. FIG. 6B is a graphshowing the dose in blood (%) as a function of time (hours) for PLA-HPGand PLA-PEG nanoparticles. FIGS. 6C and 6D are graphs showing the doseof PLA-HPG or PLA-PEG NPs per gram tissue (%) as a function of tissue at12 hours (FIG. 6C) and 24 hours (FIG. 6D). FIGS. 6E and 6F are graphsshowing the dose of PLA-HPG or PLA-PEG NPs per organ (%) as a functionof organ at 12 hours (FIG. 6E) and 24 hours (FIG. 6F).

FIG. 7A is a graph showing tumor volume (mm³) as a function of timeafter tumor inoculation (days) for PLA-HPG nanoparticles, PBS,PLA-PEG/CPT nanoparticles, PLA-HPG/CPT nanoparticles, and free CPT. FIG.7B is a graph showing the weight of mice (grams) as a function of timeafter tumor inoculation (days) for PLA-HPG nanoparticles, PBS,PLA-PEG/CPT nanoparticles, PLA-HPG/CPT nanoparticles, and free CPT.

FIG. 8 is a graph showing percent dose in blood (%) as a function oftime (hours) for PLA-HPG (PH), PLA-PEG (PP), PLA-HPG_(ALD) (PA), andPLA-HPG_(Reversed) (PLA-HPG NPs reversed from PLA-HPG_(ALD) NPs) (PR)nanoparticles.

FIG. 9A is a graph showing the number of aldehyde groups/nm² on stealthNPs as a function of incubation time with NaIO₄. Data are shown asmean±SD (n=4). FIG. 9B is a graph showing surface immobilization of DiDloaded PLA-HPG NPs treated with NaIO₄ for different period of time onlysine coated slides. Data are shown as mean±SD (n=4). FIG. 9C is agraph if relative fluorescence (%) obtained from sections of umbilicalcord incubated with DiD-loaded PLA-HPG_(ALD) and PLA-HPG NPs at 1 mg/mlon the lumen side of umbilical vein for 2 hours in Ringer's buffer at37° C. The fluorescence was quantified and normalized to the averagefluorescence of the PLA-HPG_(ALD) on umbilical vein. Data are shown asmean±SD (n=6).

FIG. 10A is a diagram of PLA-HPG_(ALD) NPs adhered to the surface ofabdominal tissues and PLA-HPG NPs diffused to all abdominal cavity andremoved by lymphatic drainage. FIG. 10B is a diagram of metastasizedtumor cells attached to the surface of abdominal tissues. FIG. 10C is adiagram showing tumor cell growth is suppressed by PLA-HPG_(ALD)/EB NPson the abdominal tissues.

FIG. 11A is a graph of drug release in (%) from EB/PLA-HPG_(ALD) NPs(EB/BNPs) and EB/PLA-HPG NPs (EB/NNPs) over time. Data are shown as meanSD (n=4). FIG. 11B is a graph of cell viability (%) of USPC cells afterincubation with free EB, EB/PLA-HPG NPs (EB/NNPs) and EB/PLA-HPG_(ALD)NPs (EB/BNPs) for 3 days. Data are shown as mean±SD (n=8). FIG. 11C is agraph of cell viability (%) of USPC cells after incubation with blankPLA-HPG_(ALD) NPs (BNPs) and blank PLA-HPG NPs (NNPs) for 3 days. Dataare shown as mean±SD (n=8). FIG. 11D is a graph of cell viability (%) ofHela and HUVEC cells after incubation with blank PLA-HPG_(ALD) NPs(BNPs) and blank PLA-HPG NPs (NNPs) for 3 days. Data are shown asmean±SD (n=8). FIG. 11E is a graph of cell viability (%) of multiplecell lines after incubation with blank PLA-HPG_(ALD) NPs (BNPs) andblank PLA-HPG NPs (NNPs) for 3 days. Data are shown as mean±SD (n=8).

FIG. 12 is a graph of the percentage surface density (%) of USPC cellsattached to slides coated with EB/PLA-HPG_(ALD) NPs (EB/BNPs),PLA-HPG_(ALD) NPs (BNPs), EB/PLA-HPG NPs (EB/NNPs), or PBS (Control).The surface density of cells was normalized to the PBS control. Data areshown as mean±SD (n=3).

FIG. 13 is a Kaplan Meier survival curve of mice bearing intraperitonealUSPC tumors and treated with intraperitoneal injection of PBS (1),PLA-HPG_(ALD) NPs in PBS (BNPs, (2)), free EB (3), EB/PLA-HPG NPs in PBS(EB/NNPs, (4)), and EB/PLA-HPG_(ALD) NPs in PBS (EB/BNPs, (5)).

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

“Effective amount” or “therapeutically effective amount”, as usedherein, refers to an amount of drug effective to alleviate, delay onsetof, or prevent one or more symptoms of a disease or disorder.

The terms “treating” or “preventing”, as used herein, can includepreventing a disease, disorder or condition from occurring in an animalwhich may be predisposed to the disease, disorder and/or condition buthas not yet been diagnosed as having it; inhibiting the disease,disorder or condition, e.g., impeding its progress; and relieving thedisease, disorder, or condition, e.g., causing regression of thedisease, disorder and/or condition. Treating the disease, disorder, orcondition can include ameliorating at least one symptom of theparticular disease, disorder, or condition, even if the underlyingpathophysiology is not affected, such as treating the pain of a subjectby administration of an analgesic agent even though such agent does nottreat the cause of the pain.

“Parenteral administration”, as used herein, means administration by anymethod other than through the digestive tract or non-invasive topical orregional routes. For example, parenteral administration may includeadministration to a patient intravenously, intradermally,intraperitoneally, intrapleurally, intratracheally, intramuscularly,subcutaneously, subjunctivally, by injection, and by infusion.

“Enteral administration”, as used herein, means administration viaabsorption through the gastrointestinal tract. Enteral administrationcan include oral and sublingual administration, gastric administration,or rectal administration.

“Pulmonary administration”, as used herein, means administration intothe lungs by inhalation or endotracheal administration. As used herein,the term “inhalation” refers to intake of air to the alveoli. The intakeof air can occur through the mouth or nose.

The terms “bioactive agent” and “active agent”, as used interchangeablyherein, include, without limitation, physiologically orpharmacologically active substances that act locally or systemically inthe body. A bioactive agent is a substance used for the treatment (e.g.,therapeutic agent), prevention (e.g., prophylactic agent), diagnosis(e.g., diagnostic agent), cure or mitigation of disease or illness, asubstance which affects the structure or function of the body, orpro-drugs, which become biologically active or more active after theyhave been placed in a predetermined physiological environment.

“Biocompatible” and “biologically compatible”, as used herein, generallyrefer to materials that are, along with any metabolites or degradationproducts thereof, generally non-toxic to the recipient, and do not causeany significant adverse effects to the recipient. Generally speaking,biocompatible materials are materials which do not elicit a significantinflammatory or immune response when administered to a patient.

The term “biodegradable” as used herein, generally refers to a materialthat will degrade or erode under physiologic conditions to smaller unitsor chemical species that are capable of being metabolized, eliminated,or excreted by the subject. The degradation time is a function ofcomposition and morphology. Degradation times can be from hours toweeks.

The term “pharmaceutically acceptable”, as used herein, refers tocompounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problems or complicationscommensurate with a reasonable benefit/risk ratio, in accordance withthe guidelines of agencies such as the Food and Drug Administration. A“pharmaceutically acceptable carrier”, as used herein, refers to allcomponents of a pharmaceutical formulation which facilitate the deliveryof the composition in vivo. Pharmaceutically acceptable carriersinclude, but are not limited to, diluents, preservatives, binders,lubricants, disintegrators, swelling agents, fillers, stabilizers, andcombinations thereof.

The term “molecular weight”, as used herein, generally refers to themass or average mass of a material. If a polymer or oligomer, themolecular weight can refer to the relative average chain length orrelative chain mass of the bulk polymer. In practice, the molecularweight of polymers and oligomers can be estimated or characterized invarious ways including gel permeation chromatography (GPC) or capillaryviscometry. GPC molecular weights are reported as the weight-averagemolecular weight (M_(w)) as opposed to the number-average molecularweight (M_(n)). Capillary viscometry provides estimates of molecularweight as the inherent viscosity determined from a dilute polymersolution using a particular set of concentration, temperature, andsolvent conditions.

The term “small molecule”, as used herein, generally refers to anorganic molecule that is less than about 2000 g/mol in molecular weight,less than about 1500 g/mol, less than about 1000 g/mol, less than about800 g/mol, or less than about 500 g/mol. Small molecules arenon-polymeric and/or non-oligomeric.

The term “copolymer” as used herein, generally refers to a singlepolymeric material that is comprised of two or more different monomers.The copolymer can be of any form, such as random, block, graft, etc. Thecopolymers can have any end-group, including capped or acid end groups.

“Hydrophilic,” as used herein, refers to the property of having affinityfor water. For example, hydrophilic polymers (or hydrophilic polymersegments) are polymers (or polymer segments) which are primarily solublein aqueous solutions and/or have a tendency to absorb water. In general,the more hydrophilic a polymer is, the more that polymer tends todissolve in, mix with, or be wetted by water.

“Hydrophobic,” as used herein, refers to the property of lackingaffinity for, or even repelling water. For example, the more hydrophobica polymer (or polymer segment), the more that polymer (or polymersegment) tends to not dissolve in, not mix with, or not be wetted bywater.

Hydrophilicity and hydrophobicity can be spoken of in relative terms,such as, but not limited to, a spectrum of hydrophilicity/hydrophobicitywithin a group of polymers or polymer segments. In some embodimentswherein two or more polymers are being discussed, the term “hydrophobicpolymer” can be defined based on the polymer's relative hydrophobicitywhen compared to another, more hydrophilic polymer.

The term “lipophilic”, as used herein, refers to compounds having anaffinity for lipids.

The term “amphiphilic”, as used herein, refers to a molecule combininghydrophilic and lipophilic (hydrophobic) properties.

“Nanoparticle”, as used herein, generally refers to a particle having adiameter, such as an average diameter, from about 10 nm up to but notincluding about 1 micron, preferably from 100 nm to about 1 micron. Theparticles can have any shape. Nanoparticles having a spherical shape aregenerally referred to as “nanospheres”.

“Microparticle”, as used herein, generally refers to a particle having adiameter, such as an average diameter, from about 1 micron to about 100microns, preferably from about 1 to about 50 microns, more preferablyfrom about 1 to about 30 microns, most preferably from about 1 micron toabout 10 microns. The microparticles can have any shape. Microparticleshaving a spherical shape are generally referred to as “microspheres”.

“Mean particle size” as used herein, generally refers to the statisticalmean particle size (diameter) of the particles in a population ofparticles. The diameter of an essentially spherical particle may referto the physical or hydrodynamic diameter. The diameter of anon-spherical particle may refer preferentially to the hydrodynamicdiameter. As used herein, the diameter of a non-spherical particle mayrefer to the largest linear distance between two points on the surfaceof the particle. Mean particle size can be measured using methods knownin the art, such as dynamic light scattering.

“Monodisperse” and “homogeneous size distribution”, are usedinterchangeably herein and describe a population of nanoparticles ormicroparticles where all of the particles are the same or nearly thesame size. As used herein, a monodisperse distribution refers toparticle distributions in which 90% or more of the distribution lieswithin 15% of the median particle size, more preferably within 10% ofthe median particle size, most preferably within 5% of the medianparticle size.

“Pharmaceutically acceptable”, as used herein, refers to compounds,carriers, excipients, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

“Branch point”, as used herein, refers to a portion of a polymer-drugconjugate that serves to connect one or more hydrophilic polymersegments to one or more hydrophobic polymer segments.

“Implant,” as generally used herein, refers to a polymeric device orelement that is structured, sized, or otherwise configured to beimplanted, preferably by injection or surgical implantation, in aspecific region of the body so as to provide therapeutic benefit byreleasing one or more agents over an extended period of time at the siteof implantation.

The term “targeting moiety”, as used herein, refers to a moiety thatbinds to or localizes to a specific locale. The moiety may be, forexample, a protein, nucleic acid, nucleic acid analog, carbohydrate, orsmall molecule. The locale may be a tissue, a particular cell type, or asubcellular compartment. The targeting moiety or a sufficient pluralityof targeting moieties may be used to direct the localization of aparticle or an active entity. The active entity may be useful fortherapeutic, prophylactic, or diagnostic purposes.

The term “reactive coupling group”, as used herein, refers to anychemical functional group capable of reacting with a second functionalgroup to form a covalent bond. The selection of reactive coupling groupsis within the ability of the skilled artisan. Examples of reactivecoupling groups can include primary amines (—NH₂) and amine-reactivelinking groups such as isothiocyanates, isocyanates, acyl azides, NHSesters, sulfonyl chlorides, aldehydes, glyoxals, epoxides, oxiranes,carbonates, aryl halides, imidoesters, carbodiimides, anhydrides, andfluorophenyl esters. Most of these conjugate to amines by eitheracylation or alkylation. Examples of reactive coupling groups caninclude aldehydes (—COH) and aldehyde reactive linking groups such ashydrazides, alkoxyamines, and primary amines. Examples of reactivecoupling groups can include thiol groups (—SH) and sulfhydryl reactivegroups such as maleimides, haloacetyls, and pyridyl disulfides. Examplesof reactive coupling groups can include photoreactive coupling groupssuch as aryl azides or diazirines. The coupling reaction may include theuse of a catalyst, heat, pH buffers, light, or a combination thereof.

The term “protective group”, as used herein, refers to a functionalgroup that can be added to and/or substituted for another desiredfunctional group to protect the desired functional group from certainreaction conditions and selectively removed and/or replaced to deprotector expose the desired functional group. Protective groups are known tothe skilled artisan. Suitable protective groups may include thosedescribed in Greene, T. W. and Wuts, P. G. M., Protective Groups inOrganic Synthesis, (1991). Acid sensitive protective groups includedimethoxytrityl (DMT), tert-butylcarbamate (tBoc) and trifluoroacetyl(tFA). Base sensitive protective groups include9-fluorenylmethoxycarbonyl (Fmoc), isobutyrl (iBu), benzoyl (Bz) andphenoxyacetyl (pac). Other protective groups include acetamidomethyl,acetyl, tert-amyloxycarbonyl, benzyl, benzyloxycarbonyl,2-(4-biphenylyl)-2-propyloxycarbonyl, 2-bromobenzyloxycarbonyl,tert-butyl₇ tert-butyloxycarbonyl,1-carbobenzoxamido-2,2,2-trifluoroethyl, 2,6-dichlorobenzyl,2-(3,5-dimethoxyphenyl)-2-propyloxycarbonyl, 2,4-dinitrophenyl,dithiasuccinyl, formyl, 4-methoxybenzenesulfonyl, 4-methoxybenzyl,4-methylbenzyl, o-nitrophenylsulfenyl, 2-phenyl-2-propyloxycarbonyl,α-2,4,5-tetramethylbenzyloxycarbonyl, p-toluenesulfonyl, xanthenyl,benzyl ester, N-hydroxysuccinimide ester, p-nitrobenzyl ester,p-nitrophenyl ester, phenyl ester, p-nitrocarbonate,p-nitrobenzylcarbonate, trimethylsilyl and pentachlorophenyl ester.

II. Core-Shell Microparticles and Nanoparticles

A. Core

The core of the particles is formed of or contains one or morehydrophobic or more hydrophobic materials, such as one or more polymericmaterials (e.g., homopolymer, copolymer, terpolymer, etc.). The materialmay be biodegradable or non-biodegradable. In some embodiments, the oneor more materials are one or more biodegradable polymers.

In general, synthetic polymers are preferred, although natural polymersmay be used and have equivalent or even better properties, especiallysome of the natural biopolymers which degrade by hydrolysis, such assome of the polyhydroxyalkanoates. Representative synthetic polymersare: poly(hydroxy acids) such as poly(lactic acid), poly(glycolic acid),and poly(lactic acid-co-glycolic acid), poly(lactide), poly(glycolide),poly(lactide-co-glycolide), polyanhydrides, polyorthoesters, polyamides,polycarbonates, polyalkylenes such as polyethylene and polypropylene,polyalkylene glycols such as poly(ethylene glycol), polyalkylene oxidessuch as poly(ethylene oxide), polyalkylene terepthalates such aspoly(ethylene terephthalate), polyvinyl alcohols, polyvinyl ethers,polyvinyl esters, polyvinyl halides such as poly(vinyl chloride),polyvinylpyrrolidone, polysiloxanes, poly(vinyl alcohols), poly(vinylacetate), polystyrene, polyurethanes and co-polymers thereof,derivativized celluloses such as alkyl cellulose, hydroxyalkylcelluloses, cellulose ethers, cellulose esters, nitro celluloses, methylcellulose, ethyl cellulose, hydroxypropyl cellulose, hydroxy-propylmethyl cellulose, hydroxybutyl methyl cellulose, cellulose acetate,cellulose propionate, cellulose acetate butyrate, cellulose acetatephthalate, carboxylethyl cellulose, cellulose triacetate, and cellulosesulfate sodium salt (jointly referred to herein as “syntheticcelluloses”), polymers of acrylic acid, methacrylic acid or copolymersor derivatives thereof including esters, poly(methyl methacrylate),poly(ethyl methacrylate), poly(butylmethacrylate), poly(isobutylmethacrylate), poly(hexylmethacrylate), poly(isodecyl methacrylate),poly(lauryl methacrylate), poly(phenyl methacrylate), poly(methylacrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), andpoly(octadecyl acrylate) (jointly referred to herein as “polyacrylicacids”), poly(butyric acid), poly(valeric acid), andpoly(lactide-co-caprolactone), copolymers and blends thereof. As usedherein, “derivatives” include polymers having substitutions, additionsof chemical groups and other modifications routinely made by thoseskilled in the art.

Examples of preferred natural polymers include proteins such as albumin,collagen, gelatin and prolamines, for example, zein, and polysaccharidessuch as alginate, cellulose derivatives and polyhydroxyalkanoates, forexample, polyhydroxybutyrate. The in vivo stability of themicroparticles can be adjusted during the production by using polymerssuch as poly(lactide-co-glycolide) copolymerized with polyethyleneglycol (PEG). If PEG is exposed on the external surface, it may increasethe time these materials circulate due to the hydrophilicity of PEG.

Examples of preferred non-biodegradable polymers include ethylene vinylacetate, poly(meth)acrylic acid, polyamides, copolymers and mixturesthereof.

In certain embodiments, the hydrophobic polymer is an aliphaticpolyester. In preferred embodiments, the hydrophobic polymer ispoly(lactic acid), poly(glycolic acid), or poly(lactic acid-co-glycolicacid).

The particles are designed to release molecules to be encapsulated orattached over a period of days to weeks. Factors that affect theduration of release include pH of the surrounding medium (higher rate ofrelease at pH 5 and below due to acid catalyzed hydrolysis of PLGA) andpolymer composition. Aliphatic polyesters differ in hydrophobicity andthat in turn affects the degradation rate. The hydrophobic poly (lacticacid) (PLA), more hydrophilic poly (glycolic acid) PGA and theircopolymers, poly (lactide-co-glycolide) (PLGA) have various releaserates. The degradation rate of these polymers, and often thecorresponding drug release rate, can vary from days (PGA) to months(PLA) and is easily manipulated by varying the ratio of PLA to PGA.

The core can be formed of copolymers including amphiphilic copolymerssuch as PLGA-PEG or PLURONICS (block copolymers of polyethyleneoxide-polypropylene glycol) but this may decrease the benefit of thepolyglycerol molecules discussed below.

Other materials may also be incorporated including lipids, fatty acids,and phospholipids. These may be dispersed in or on the particles, orinterspersed with the polyglycerol coatings discussed below.

B. Shell

The particles described herein contain a shell or coating containinghyperbranched polyglycerol (HPG).

Hyperbranched polyglycerol is a highly branched polyol containing apolyether scaffold. Hyperbranched polyglycerol can be prepared usingtechniques known in the art. It can be formed from controlledetherification of glycerol via cationic or anionic ring openingmultibranching polymerization of glycidol. For example, an initiatorhaving multiple reactive sites is reacted with glycidol in the presenceof a base to form hyperbranched polyglycerol (HPG). Suitable initiatorsinclude, but are not limited to, polyols, e.g., triols, tetraols,pentaols, or greater and polyamines, e.g., triamines, tetraamines,pentaamines, etc. In one embodiment, the initiator is1,1,1-trihydroxymethyl propane (THP).

A formula for hyperbranched polyglycerol as described in EP 2754684 is

wherein o, p and q are independently integers from 1-100,wherein A₁ and A₂ are independently

wherein l, m and n are independently integers from 1-100.wherein A₃ and A₄ are defined as A₁ and A₂, with the proviso that A₃ andA₄ are hydrogen, n and m are each 1 for terminal residues.

The surface properties of the HPG can be tuned based on the chemistry ofvicinal diols. For example, the surface properties can be tuned toprovide stealth particles, i.e., particles that are not cleared by theMPS due to the presence of the hydroxyl groups; adhesive (sticky)particles, i.e., particles that adhere to the surface of tissues, forexample, due to the presence of one or more reactive functional groups,such as aldehydes, amines, oxime, or O-substituted oxime that can beprepared from the vicinal hydroxyl moieties; or targeting by theintroduction of one or more targeting moieties which can be conjugateddirectly or indirectly to the vicinal hydroxyl moieties. Indirectlyrefers to transformation of the hydroxy groups to reactive functionalgroups that can react with functional groups on molecules to be attachedto the surface, such as active agents and/or targeting moieties, etc. Aschematic of this tunability is shown in FIG. 1.

The hyperbranched nature of the polyglycerol allows for a much higherdensity of hydroxyl groups, reactive functional groups, and/or targetingmoieties than polyethylene glycol. For example, the particles describedherein can have a density of surface functionality (e.g., hydroxylgroups, reactive functional groups, and/or targeting moieties) of atleast about 1, 2, 3, 4, 5, 6, 7, or 8 groups/nm².

The molecular weight of the HPG can vary. For example, in thoseembodiments wherein the HPG is covalently attached to the materials orpolymers that form the core, the molecular weight can vary depending onthe molecular weight and/or hydrophobicity of the core materials. Themolecular weight of the HPG is generally from about 1,000 to about1,000,000 Daltons, from about 1,000 to about 500,000 Daltons, from about1,000 to about 250,000 Daltons, or from about 1,000 to about 100,000Daltons. In those embodiments wherein the HPG is covalently bound to thecore materials, the weight percent of HPG of the copolymer is from about1% to about 50%, such as about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35,40, 45 or 50%.

In some embodiments, the HPG is covalently coupled to a hydrophobicmaterial or a more hydrophobic material, such as a polymer. Uponself-assembly, particles are formed containing a core containing thehydrophobic material and a shell or coating of HPG. HPG coupled to thepolymer PLA is shown below:

C. Molecules to be Encapsulated or Attached to the Surface of theParticles

The particles described herein may contain one or more moleculesencapsulated within and/or attached to the surface of the particles. Themolecules can be covalently or non-covalently associated with theparticles. In some embodiments, the molecules are targeting moietieswhich are covalently associated with the particles. In particularembodiments, the targeting moieties are covalently bound to the HPGcoating via the hydroxy groups on HPG. The targeting moieties can bebound directly to HPG or via a coupling agent. In other embodiments, theparticles have encapsulated therein one or more therapeutic agents,diagnostic agents, prophylactic agents, and/or nutraceuticals. In someembodiments, the particles contain both targeting agents which arecovalently or non-covalently associated with the particles and one ormore therapeutic agents, diagnostic agents, prophylactic agents, and/ornutraceuticals which are covalently or non-covalently associated withthe particles.

1. Covalently Bound Molecules

Molecules can be bound to the hydroxy groups on HPG before or afterparticle formation. Representative methodologies for conjugatedmolecules to the hydroxy groups on HPG are described below.

One useful protocol involves the “activation” of hydroxyl groups withcarbonyldiimidazole (CDI) in aprotic solvents such as DMSO, acetone, orTHF. CDI forms an imidazolyl carbamate complex with the hydroxyl groupwhich may be displaced by binding the free amino group of a ligand suchas a protein. The reaction is an N-nucleophilic substitution and resultsin a stable N-alkylcarbamate linkage of the ligand to the polymer. The“coupling” of the ligand to the “activated” polymer matrix is maximal inthe pH range of 9-10 and normally requires at least 24 hrs. Theresulting ligand-polymer complex is stable and resists hydrolysis forextended periods of time.

Another coupling method involves the use of1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC) or “water-solubleCDI” in conjunction with N-hydroxylsulfosuccinimide (sulfo NHS) tocouple the exposed carboxylic groups of polymers to the free aminogroups of ligands in a totally aqueous environment at the physiologicalpH of 7.0. Briefly, EDAC and sulfo-NHS form an activated ester with thecarboxylic acid groups of the polymer which react with the amine end ofa ligand to form a peptide bond. The resulting peptide bond is resistantto hydrolysis. The use of sulfo-NHS in the reaction increases theefficiency of the EDAC coupling by a factor of ten-fold and provides forexceptionally gentle conditions that ensure the viability of theligand-polymer complex.

By using either of these protocols it is possible to “activate” almostall polymers containing either hydroxyl or carboxyl groups in a suitablesolvent system that will not dissolve the polymer matrix.

A useful coupling procedure for attaching ligands with free hydroxyl andcarboxyl groups to polymers involves the use of the cross-linking agent,divinylsulfone. This method would be useful for attaching sugars orother hydroxylic compounds with bioadhesive properties to hydroxylicmatrices. Briefly, the activation involves the reaction ofdivinylsulfone to the hydroxyl groups of the polymer, forming thevinylsulfonyl ethyl ether of the polymer. The vinyl groups will coupleto alcohols, phenols and even amines. Activation and coupling take placeat pH 11. The linkage is stable in the pH range from 1-8 and is suitablefor transit through the intestine.

Alternatively, the hydroxyl groups can be converted to reactivefunctional group that can react with a reactive functional group on themolecule to be attached. For example, the hydroxyl groups on HPG can beconverted to aldehydes, amines, or O-substituted oximes which can reactwith reactive functional groups on molecules to be attached. Suchtransformations can be done before or after particle formation.

Any suitable coupling method known to those skilled in the art for thecoupling of ligands and polymers with double bonds, including the use ofUV crosslinking, may be used for attachment of molecules to the polymer.

Coupling is preferably by covalent binding but it may also be indirect,for example, through a linker bound to the polymer or through aninteraction between two molecules such as strepavidin and biotin. It mayalso be by electrostatic attraction by dip-coating.

The coupling methods can be done before or after particle formation.

2. Therapeutic Agent, Diagnostic Agents, Prophylactic Agents, and/orNutraceuticals

Agents to be delivered include therapeutic, nutritional, diagnostic, andprophylactic compounds. Proteins, peptides, carbohydrates,polysaccharides, nucleic acid molecules, and organic molecules, as wellas diagnostic agents, can be delivered. The preferred materials to beincorporated are drugs and imaging agents. Therapeutic agents includeantibiotics, antivirals, anti-parasites (helminths, protozoans),anti-cancer (referred to herein as “chemotherapeutics”, includingcytotoxic drugs such as doxorubicin, cyclosporine, mitomycin C,cisplatin and carboplatin, BCNU, 5FU, methotrexate, adriamycin,camptothecin, epothilones A-F, and taxol), antibodies and bioactivefragments thereof (including humanized, single chain, and chimericantibodies), antigen and vaccine formulations, peptide drugs,anti-inflammatories, nutraceuticals such as vitamins, andoligonucleotide drugs (including DNA, RNAs, antisense, aptamers,ribozymes, external guide sequences for ribonuclease P, and triplexforming agents).

Particularly preferred drugs to be delivered include anti-angiogenicagents, antiproliferative and chemotherapeutic agents such asrampamycin. Incorporated into microparticles, these agents may be usedto treat cancer or eye diseases, or prevent restenosis followingadministration into the blood vessels.

Representative classes of diagnostic materials include paramagneticmolecules, fluorescent compounds, magnetic molecules, and radionuclides.Exemplary materials include, but are not limited to, metal oxides, suchas iron oxide, metallic particles, such as gold particles, etc.Biomarkers can also be conjugated to the surface for diagnosticapplications.

One or more active agents may be formulated alone or with excipients orencapsulated on, in or incorporated into the microparticles ornanoparticles. Active agents include therapeutic, prophylactic,neutraceutical and diagnostic agents. Any suitable agent may be used.These include organic compounds, inorganic compounds, proteins,polysaccharides, nucleic acids or other materials that can beincorporated using standard techniques.

Active agents include synthetic and natural proteins (including enzymes,peptide-hormones, receptors, growth factors, antibodies, signalingmolecules), and synthetic and natural nucleic acids (including RNA, DNA,anti-sense RNA, triplex DNA, inhibitory RNA (RNAi), andoligonucleotides), and biologically active portions thereof. Suitableactive agents have a size greater than about 1,000 Da for small peptidesand polypeptides, more typically at least about 5,000 Da and often10,000 Da or more for proteins. Nucleic acids are more typically listedin terms of base pairs or bases (collectively “bp”). Nucleic acids withlengths above about 10 bp are typically used in the present method. Moretypically, useful lengths of nucleic acids for probing or therapeuticuse will be in the range from about 20 bp (probes; inhibitory RNAs,etc.) to tens of thousands of bp for genes and vectors. The activeagents may also be hydrophilic molecules, preferably having a lowmolecular weight.

Examples of useful proteins include hormones such as insulin and growthhormones including somatomedins Examples of useful drugs includeneurotransmitters such as L-DOPA, antihypertensives or saluretics suchas Metolazone from Searle Pharmaceuticals, carbonic anhydrase inhibitorssuch as Acetazolamide from Lederle Pharmaceuticals, insulin like drugssuch as glyburide, a blood glucose lowering drug of the sulfonylureaclass, synthetic hormones such as Android F from Brown Pharmaceuticalsand Testred® (methyltestosterone) from ICN Pharmaceuticals.

Representative anti-cancer agents include, but are not limited to,alkylating agents (such as cisplatin, carboplatin, oxaliplatin,mechlorethamine, cyclophosphamide, chlorambucil, dacarbazine, lomustine,carmustine, procarbazine, chlorambucil and ifosfamide), antimetabolites(such as fluorouracil (5-FU), gemcitabine, methotrexate, cytosinearabinoside, fludarabine, and floxuridine), antimitotics (includingtaxanes such as paclitaxel and decetaxel, epothilones A-F, and vincaalkaloids such as vincristine, vinblastine, vinorelbine, and vindesine),anthracyclines (including doxorubicin, daunorubicin, valrubicin,idarubicin, and epirubicin, as well as actinomycins such as actinomycinD), cytotoxic antibiotics (including mitomycin, plicamycin, andbleomycin), topoisomerase inhibitors (including camptothecins such ascamptothecin, irinotecan, and topotecan as well as derivatives ofepipodophyllotoxins such as amsacrine, etoposide, etoposide phosphate,and teniposide), and combinations thereof. Other suitable anti-canceragents include angiogenesis inhibitors including antibodies to vascularendothelial growth factor (VEGF) such as bevacizumab (AVASTIN®), otheranti-VEGF compounds; thalidomide (THALOMID®) and derivatives thereofsuch as lenalidomide (REVLIMID®); endostatin; angiostatin; receptortyrosine kinase (RTK) inhibitors such as sunitinib (SUTENT®); tyrosinekinase inhibitors such as sorafenib (Nexavar®), erlotinib (Tarceva®),pazopanib, axitinib, and lapatinib; transforming growth factor-α ortransforming growth factor-β inhibitors, and antibodies to the epidermalgrowth factor receptor such as panitumumab (VECTIBIX®) and cetuximab(ERBITUX®).

Under the Biopharmaceutical Classification System (BCS), drugs canbelong to four classes: class I (high permeability, high solubility),class II (high permeability, low solubility), class III (lowpermeability, high solubility) or class IV (low permeability, lowsolubility). Suitable active agents also include poorly solublecompounds; such as drugs that are classified as class II or class IVcompounds using the BCS. Examples of class II compounds include:acyclovir, nifedipine, danazol, ketoconazole, mefenamic acid,nisoldipine, nicardipine, felodipine, atovaquone, griseofulvin,troglitazone glibenclamide and carbamazepine. Examples of class IVcompounds include: chlorothiazide, furosemide, tobramycin, cefuroxmine,and paclitaxel.

For imaging, radioactive materials such as Technetium99 (^(99m)Tc) ormagnetic materials such as Fe₂O₃ could be used. Examples of othermaterials include gases or gas emitting compounds, which areradioopaque.

Alternatively, the biodegradable polymers may encapsulate cellularmaterials, such as for example, cellular materials to be delivered toantigen presenting cells as described below to induce immunologicalresponses.

Peptide, protein, and DNA based vaccines may be used to induce immunityto various diseases or conditions. For example, sexually transmitteddiseases and unwanted pregnancy are world-wide problems affecting thehealth and welfare of women. Effective vaccines to induce specificimmunity within the female genital tract could greatly reduce the riskof STDs, while vaccines that provoke anti-sperm antibodies wouldfunction as immunocontraceptives. Extensive studies have demonstratedthat vaccination at a distal site—orally, nasally, or rectally, forexample—can induce mucosal immunity within the female genital tract. Ofthese options, oral administration has gained the most interest becauseof its potential for patient compliance, easy administration andsuitability for widespread use. Oral vaccination with proteins ispossible, but is usually inefficient or requires very high doses. Oralvaccination with DNA, while potentially effective at lower doses, hasbeen ineffective in most cases because ‘naked DNA’ is susceptible toboth the stomach acidity and digestive enzymes in the gastrointestinaltract

Cell-mediated immunity is needed to detect and destroy virus-infectedcells. Most traditional vaccines (e.g. protein-based vaccines) can onlyinduce humoral immunity. DNA-based vaccine represents a unique means tovaccinate against a virus or parasite because a DNA based vaccine caninduce both humoral and cell-mediated immunity. In addition, DNA-basedvaccines are potentially safer than traditional vaccines. DNA vaccinesare relatively more stable and more cost-effective for manufacturing andstorage. DNA vaccines consist of two major components—DNA carriers (ordelivery vehicles) and DNAs encoding antigens. DNA carriers protect DNAfrom degradation, and can facilitate DNA entry to specific tissues orcells and expression at an efficient level.

Biodegradable polymer particles offer several advantages for use as DNAdelivery vehicles for DNA based vaccines. The polymer particles can bebiodegradable and biocompatible, and they have been used successfully inpast therapeutic applications to induce mucosal or humoral immuneresponses. Polymer biodegradation products are typically formed at arelatively slow rate, are biologically compatible, and result inmetabolizable moieties. Biodegradable polymer particles can bemanufactured at sizes ranging from diameters of several microns(microparticles) to particles having diameters of less than one micron(nanoparticles).

Dendritic cells (DCs) are recognized to be powerful antigen presentingcells for inducing cellular immunologic responses in humans. DCs primeboth CD8+ cytotoxic T-cell (CTL) and CD4+ T-helper (Th1) responses. DCsare capable of capturing and processing antigens, and migrating to theregional lymph nodes to present the captured antigens and induce T-cellresponses. Immature DCs can internalize and process cellular materials,such as DNA encoding antigens, and induce cellular immunologic responsesto disease effectors.

As used herein, the term “disease effector agents” refers to agents thatare central to the causation of a disease state in a subject. In certaincircumstances, these disease effector agents are disease-causing cellswhich may be circulating in the bloodstream, thereby making them readilyaccessible to extracorporeal manipulations and treatments. Examples ofsuch disease-causing cells include malignant T cells, malignant B cells,T cells and B cells which mediate an autoimmune response, and virally orbacterially infected white blood cells which express on their surfaceviral or bacterial peptides or proteins. Exemplary disease categoriesgiving rise to disease-causing cells include leukemia, lymphoma,autoimmune disease, graft versus host disease, and tissue rejection.Disease associated antigens which mediate these disease states and whichare derived from disease-causing cells include peptides that bind to aMHC Class I site, a MHC Class II site, or to a heat shock protein whichis involved in transporting peptides to and from MHC sites (i.e., achaperone). Disease associated antigens also include viral or bacterialpeptides which are expressed on the surface of infected white bloodcells, usually in association with an MHC Class I or Class II molecule.

Other disease-causing cells include those isolated from surgicallyexcised specimens from solid tumors, such as lung, colon, brain, kidneyor skin cancers. These cells can be manipulated extracorporeally inanalogous fashion to blood leukocytes, after they are brought intosuspension or propagated in tissue culture. Alternatively, in someinstances, it has been shown that the circulating blood of patients withsolid tumors can contain malignant cells that have broken off from thetumors and entered the circulation. These circulating tumor cells canprovide an easily accessible source of cancer cells which may berendered apoptotic and presented to the antigen presenting cells.

In addition to disease-causing cells, disease effector agents includemicrobes such as bacteria, fungi, yeast, viruses which express or encodedisease-associated antigens, and prions.

The disease effector agents are presented to the antigen presentingcells using biodegradable polymer microparticles as delivery vehicles.The loaded microparticles are exposed to immature antigen presentingcells, which internalize the microparticles and process the materialwithin the microparticles. The microparticles may be administered to thepatient and the interaction between the microparticles and the antigenpresenting cells may occur in vivo. In a preferred embodiment, themicroparticles are placed in an incubation bag with the immature antigenpresenting cells, and the microparticles are phagocytosed by the antigenpresenting cells during the incubation period. The resulting antigenpresenting cells are then administered to the patient to induce animmune response to the disease causing agent.

Other agents include cell penetrating peptides, such as TAT,Antennapedia, polyarginine and poly-lysine analogues.

3. Targeting Moieties

The particles, such as the surface of the particles, can be modified tofacilitate targeting through the attachment of targeting molecules.Exemplary target molecules include proteins, peptides, nucleic acids,lipids, saccharides, or polysaccharides, or small molecules that bind toone or more targets associated with an organ, tissue, cell, orextracellular matrix, or specific type of tumor or infected cell. Thedegree of specificity with which the particles are targeted can bemodulated through the selection of a targeting molecule with theappropriate affinity and specificity. For example, a targeting moietycan be a polypeptide, such as an antibody that specifically recognizes atumor marker that is present exclusively or in higher amounts on amalignant cell (e.g., a tumor antigen). Suitable targeting moleculesthat can be used to direct nanoparticles to cells and tissues ofinterest, as well as methods of conjugating target molecules tonanoparticles, are known in the art. See, for example, Ruoslahti, et al.Nat. Rev. Cancer, 2:83-90 (2002). Targeting molecules can also includeneuropilins and endothelial targeting molecules, integrins, selectins,and adhesion molecules.

Targeting molecules can be covalently bound to particles using a varietyof methods known in the art. In some embodiments, the targeting moietiesare covalently associated with the polymer, preferably via a linkercleaved at the site of delivery.

The nanoparticles can contain one or more polymer conjugates containingend-to-end linkages between the polymer and a targeting element or adetectable label. For example, a modified polymer can be aPLA-HPG-peptide block polymer.

Examples of targeting moieties include peptides such as iRGD, LyP1;small molecule such as folate, aptamers and antibodies or theircombinations at various molar ratios.

The targeting element of the nanoparticle can be an antibody or antigenbinding fragment thereof. The targeting elements should have an affinityfor a cell-surface receptor or cell-surface antigen on the target cellsand result in internalization of the particle within the target cell.

The targeting element can specifically recognize and bind to a targetmolecule specific for a cell type, a tissue type, or an organ. Thetarget molecule can be a cell surface polypeptide, lipid, or glycolipid.The target molecule can be a receptor that is selectively expressed on aspecific cell surface, a tissue or an organ. Cell specific markers canbe for specific types of cells including, but not limited to stem cells,blood cells, immune cells, muscle cells, nerve cells, cancer cells,virally infected cells, and organ specific cells. The cell markers canbe specific for endothelial, ectodermal, or mesenchymal cells.Representative cell specific markers include, but are not limited tocancer specific markers.

Additional targets that can be recognized by the targeting elementinclude VEGF/KDR, Tie2, vascular cell adhesion molecule (VCAM), endoglinand α₅β₃ integrin/vitronectin. The targeting peptides can be covalentlyassociated with the polymer of the outer shell and the covalentassociation can be mediated by a linker.

Tumor-Specific and Tumor-Associated Antigens

In one embodiment the targeting element specifically binds to an antigenthat is expressed by tumor cells. The antigen expressed by the tumor maybe specific to the tumor, or may be expressed at a higher level on thetumor cells as compared to non-tumor cells. Antigenic markers such asserologically defined markers known as tumor associated antigens, whichare either uniquely expressed by cancer cells or are present at markedlyhigher levels (e.g., elevated in a statistically significant manner) insubjects having a malignant condition relative to appropriate controls,are contemplated for use in certain embodiments.

Tumor-associated antigens may include, for example, cellularoncogene-encoded products or aberrantly expressed proto-oncogene-encodedproducts (e.g., products encoded by the neu, ras, trk, and kit genes),or mutated forms of growth factor receptor or receptor-like cell surfacemolecules (e.g., surface receptor encoded by the c-erb B gene). Othertumor-associated antigens include molecules that may be directlyinvolved in transformation events, or molecules that may not be directlyinvolved in oncogenic transformation events but are expressed by tumorcells (e.g., carcinoembryonic antigen, CA-125, melonoma associatedantigens, etc.) (see, e.g., U.S. Pat. No. 6,699,475; Jager, et al., Int.J. Cancer, 106:817-20 (2003); Kennedy, et al., Int. Rev. Immunol.,22:141-72 (2003); Scanlan, et al. Cancer Immun., 4:1 (2004)).

Genes that encode cellular tumor associated antigens include cellularoncogenes and proto-oncogenes that are aberrantly expressed. In general,cellular oncogenes encode products that are directly relevant to thetransformation of the cell, and because of this, these antigens areparticularly preferred targets for immunotherapy. An example is thetumorigenic neu gene that encodes a cell surface molecule involved inoncogenic transformation. Other examples include the ras, kit, and trkgenes. The products of proto-oncogenes (the normal genes which aremutated to form oncogenes) may be aberrantly expressed (e.g.,overexpressed), and this aberrant expression can be related to cellulartransformation. Thus, the product encoded by proto-oncogenes can betargeted. Some oncogenes encode growth factor receptor molecules orgrowth factor receptor-like molecules that are expressed on the tumorcell surface. An example is the cell surface receptor encoded by thec-erbB gene. Other tumor-associated antigens may or may not be directlyinvolved in malignant transformation. These antigens, however, areexpressed by certain tumor cells and may therefore provide effectivetargets. Some examples are carcinoembryonic antigen (CEA), CA 125(associated with ovarian carcinoma), and melanoma specific antigens.

In ovarian and other carcinomas, for example, tumor associated antigensare detectable in samples of readily obtained biological fluids such asserum or mucosal secretions. One such marker is CA125, a carcinomaassociated antigen that is also shed into the bloodstream, where it isdetectable in serum (e.g., Bast, et al., N. Eng. J. Med., 309:883(1983); Lloyd, et al., Int. J. Canc., 71:842 (1997). CA125 levels inserum and other biological fluids have been measured along with levelsof other markers, for example, carcinoembryonic antigen (CEA), squamouscell carcinoma antigen (SCC), tissue polypeptide specific antigen (TPS),sialyl TN mucin (STN), and placental alkaline phosphatase (PLAP), inefforts to provide diagnostic and/or prognostic profiles of ovarian andother carcinomas (e.g., Sarandakou, et al., Acta Oncol., 36:755 (1997);Sarandakou, et al., Eur. J Gynaecol. Oncol., 19:73 (1998); Meier, etal., Anticancer Res., 17(4B):2945 (1997); Kudoh, et al., Gynecol.Obstet. Invest., 47:52 (1999)). Elevated serum CA125 may also accompanyneuroblastoma (e.g., Hirokawa, et al., Surg. Today, 28:349 (1998), whileelevated CEA and SCC, among others, may accompany colorectal cancer(Gebauer, et al., Anticancer Res., 17(4B):2939 (1997)).

The tumor associated antigen, mesothelin, defined by reactivity withmonoclonal antibody K-1, is present on a majority of squamous cellcarcinomas including epithelial ovarian, cervical, and esophagealtumors, and on mesotheliomas (Chang, et al., Cancer Res., 52:181 (1992);Chang, et al., Int. J. Cancer, 50:373 (1992); Chang, et al., Int. J.Cancer, 51:548 (1992); Chang, et al., Proc. Natl. Acad. Sci. USA, 93:136(1996); Chowdhury, et al., Proc. Natl. Acad. Sci. USA, 95:669 (1998)).Using MAb K-1, mesothelin is detectable only as a cell-associated tumormarker and has not been found in soluble form in serum from ovariancancer patients, or in medium conditioned by OVCAR-3 cells (Chang, etal., Int. J. Cancer, 50:373 (1992)). Structurally related humanmesothelin polypeptides, however, also include tumor-associated antigenpolypeptides such as the distinct mesothelin related antigen (MRA)polypeptide, which is detectable as a naturally occurring solubleantigen in biological fluids from patients having malignancies (see WO00/50900).

A tumor antigen may include a cell surface molecule. Tumor antigens ofknown structure and having a known or described function, include thefollowing cell surface receptors: HER1 (GenBank Accession No. U48722),HER2 (Yoshino, et al., J. Immunol., 152:2393 (1994); Disis, et al.,Canc. Res., 54:16 (1994); GenBank Acc. Nos. X03363 and M17730), HER3(GenBank Acc. Nos. U29339 and M34309), HER4 (Plowman, et al., Nature,366:473 (1993); GenBank Ace. Nos. L07868 and T64105), epidermal growthfactor receptor (EGFR) (GenBank Acc. Nos. U48722, and KO3193), vascularendothelial cell growth factor (GenBank No. M32977), vascularendothelial cell growth factor receptor (GenBank Acc. Nos. AF022375,1680143, U48801 and X62568), insulin-like growth factor-I (GenBank Acc.Nos. X00173, X56774, X56773, X06043, European Patent No. GB 2241703),insulin-like growth factor-II (GenBank Acc. Nos. X03562, X00910, M17863and M17862), transferrin receptor (Trowbridge and Omary, Proc. Nat.Acad. USA, 78:3039 (1981); GenBank Acc. Nos. X01060 and M11507),estrogen receptor (GenBank Acc. Nos. M38651, X03635, X99101, U47678 andM12674), progesterone receptor (GenBank Ace. Nos. X51730, X69068 andM15716), follicle stimulating homione receptor (FSH-R) (GenBank Ace.Nos. Z34260 and M65085), retinoic acid receptor (GenBank Acc. Nos.L12060, M60909, X77664, X57280, X07282 and X06538), MUC-1 (Barnes, etal., Proc. Nat. Acad. Sci. USA, 86:7159 (1989); GenBank Acc. Nos. M65132and M64928) NY-ESO-1 (GenBank Ace. Nos. AJ003149 and U87459), NA 17-A(PCT Publication No. WO 96/40039), Melan-A/MART-1 (Kawakami, et al.,Proc. Nat. Acad. Sci. USA, 91:3515 (1994); GenBank Acc. Nos. U06654 andU06452), tyrosinase (Topalian, et al., Proc. Nat. Acad. Sci. USA,91:9461 (1994); GenBank Acc. No. M26729; Weber, et al., J. Clin. Invest,102:1258 (1998)), Gp-100 (Kawakami, et al., Proc. Nat. Acad. Sci. USA,91:3515 (1994); GenBank Acc. No. S73003, Adema, et al., J. Biol. Chem.,269:20126 (1994)), MAGE (van den Bruggen, et al., Science, 254:1643(1991)); GenBank Acc. Nos. U93163, AF064589, U66083, D32077, D32076,D32075, U10694, U10693, U10691, U10690, U10689, U10688, U10687, U10686,U10685, L18877, U10340, U10339, L18920, U03735 and M77481), BAGE(GenBank Acc. No. U19180; U.S. Pat. Nos. 5,683,886 and 5,571,711), GAGE(GenBank Acc. Nos. AF055475, AF055474, AF055473, U19147, U19146, U19145,U19144, U19143 and U19142), any of the CTA class of receptors includingin particular HOM-MEL-40 antigen encoded by the SSX2 gene (GenBank Acc.Nos. X86175, U90842, U90841 and X86174), carcinoembryonic antigen (CEA,Gold and Freedman, J. Exp. Med., 121:439 (1985); GenBank Acc. Nos.M59710, M59255 and M29540), and PyLT (GenBank Acc. Nos. J02289 andJ02038); p97 (melanotransferrin) (Brown, et al., J. Immunol., 127:539-46(1981); Rose, et al., Proc. Natl. Acad. Sci. USA, 83:1261-61 (1986)).

Additional tumor associated antigens include prostate surface antigen(PSA) (U.S. Pat. Nos. 6,677,157; 6,673,545); β-human chorionicgonadotropin β-HCG) (McManus, et al., Cancer Res., 36:3476-81 (1976);Yoshimura, et al., Cancer, 73:2745-52 (1994); Yamaguchi, et al., Br. J.Cancer, 60:382-84 (1989): Alfthan, et al., Cancer Res., 52:4628-33(1992)); glycosyltransferase β-1,4-N-acetylgalactosaminyltransferases(GalNAc) (Hoon, et al., Int. J. Cancer, 43:857-62 (1989); Ando, et al.,Int. J. Cancer, 40:12-17 (1987); Tsuchida, et al., J. Natl. Cancer,78:45-54 (1987); Tsuchida, et al., J. Natl. Cancer, 78:55-60 (1987));NUC18 (Lehmann, et al., Proc. Natl. Acad. Sci. USA, 86:9891-95 (1989);Lehmann, et al., Cancer Res., 47:841-45 (1987)); melanoma antigen gp75(Vijayasardahi, et al., J. Exp. Med., 171:1375-80 (1990); GenBankAccession No. X51455); human cytokeratin 8; high molecular weightmelanoma antigen (Natali, et al., Cancer, 59:55-63 (1987); keratin 19(Datta, et al., J. Clin. Oncol., 12:475-82 (1994)).

Tumor antigens of interest include antigens regarded in the art as“cancer/testis” (CT) antigens that are immunogenic in subjects having amalignant condition (Scanlan, et al., Cancer Immun., 4:1 (2004)). CTantigens include at least 19 different families of antigens that containone or more members and that are capable of inducing an immune response,including but not limited to MAGEA (CT1); BAGE (CT2); MAGEB (CT3); GAGE(CT4); SSX (CT5); NY-ESO-1 (CT6); MAGEC (CT7); SYCP1 (C8); SPANXB1(CT11.2); NA88 (CT18); CTAGE (CT21); SPA17 (CT22); OY-TES-1 (CT23); CAGE(CT26); HOM-TES-85 (CT28); HCA661 (CT30); NY-SAR-35 (CT38); FATE (CT43);and TPTE (CT44).

Additional tumor antigens that can be targeted, including atumor-associated or tumor-specific antigen, include, but not limited to,alpha-actinin-4, Bcr-Ab1 fusion protein, Casp-8, beta-catenin, cdc27,cdk4, cdkn2a, coa-1, dek-can fusion protein, EF2, ETV6-AML1 fusionprotein, LDLR-fucosyltransferaseAS fusion protein, HLA-A2, HLA-A11,hsp70-2, KIAAO205, Mart2, Mum-1, 2, and 3, neo-PAP, myosin class I,OS-9, pml-RARα fusion protein, PTPRK, K-ras, N-ras, Triosephosphateisomeras, Bage-1, Gage 3,4,5,6,7, GnTV, Herv-K-mel, Lage-1,Mage-A1,2,3,4,6,10,12, Mage-C2, NA-88, NY-Eso-1/Lage-2, SP17, SSX-2, andTRP2-Int2, MelanA (MART-I), gp100 (Pmel 17), tyrosinase, TRP-1, TRP-2,MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15(58), CEA, RAGE, NY-ESO (LAGE),SCP-1, Hom/Mel-40, PRAME, p53, H-Ras, HER-2/neu, BCR-ABL, E2A-PRL,H4-RET, IGH-IGK, MYL-RAR, Epstein Barr virus antigens, EBNA, humanpapillomavirus (HPV) antigens E6 and E7, TSP-180, MAGE-4, MAGE-5,MAGE-6, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG-72-4, CA 19-9,CA 72-4, CAM 17.1, NuMa, K-ras, β-Catenin, CDK4, Mum-1, p16, TAGE, PSMA,PSCA, CT7, telomerase, 43-9F, 5T4, 791Tgp72, α-fetoprotein, 13HCG,BCA225, BTAA, CA 125, CA 15-3 (CA 27.29\BCAA), CA 195, CA 242, CA-50,CAM43, CD68\KP1, CO-029, FGF-5, G250, Ga733 (EpCAM), HTgp-175, M344,MA-50, MG7-Ag, MOV18, NB\70K, NY—CO-1, RCAS1, SDCCAG16, TA-90 (Mac-2binding protein\cyclophilin C-associated protein), TAAL6, TAG72, TLP,and TPS. Other tumor-associated and tumor-specific antigens are known tothose of skill in the art and are suitable for targeting by the fusionproteins.

Peptide Targeting Elements

The targeting element can be a peptide. Specifically, the plaquetargeted peptide can be, but is not limited to, one or more of thefollowing: RGD, iRGD(CRGDK/RGPD/EC), LyP-1, P3(CKGGRAKDC), or theircombinations at various molar ratios. The targeting peptides can becovalently associated with the polymer and the covalent association canbe mediated by a linker.

Antibody Targeting Elements

The targeting element can be an antibody or an antigen-binding fragmentthereof. The antibody can be any type of immunoglobulin that is known inthe art. For instance, the antibody can be of any isotype, e.g., IgA,IgD, IgE, IgG, IgM, etc. The antibody can be monoclonal or polyclonal.The antibody can be a naturally-occurring antibody, e.g., an antibodyisolated and/or purified from a mammal, e.g., mouse, rabbit, goat,horse, chicken, hamster, human, etc. Alternatively, the antibody can bea genetically-engineered antibody, e.g., a humanized antibody or achimeric antibody. The antibody can be in monomeric or polymeric form.The antigen binding portion of the antibody can be any portion that hasat least one antigen binding site, such as Fab, F(ab′)₂, dsFv, sFv,diabodies, and triabodies. In certain embodiments, the antibody is asingle chain antibody.

Aptamer Targeting Elements

Aptamers are oligonucleotide or peptide sequences with the capacity torecognize virtually any class of target molecules with high affinity andspecificity. Aptamers bind to targets such as small organics, peptides,proteins, cells, and tissues. Unlike antibodies, some aptamers exhibitstereoselectivity. The aptamers can be designed to bind to specifictargets expressed on cells, tissues or organs.

Other Targeting Moieties

The outer surface of the microparticle may be treated using a mannoseamine, thereby mannosylating the outer surface of the microparticle.This treatment may cause the microparticle to bind to the target cell ortissue at a mannose receptor on the antigen presenting cell surface.Alternatively, surface conjugation with an immunoglobulin moleculecontaining an Fc portion (targeting Fc receptor), heat shock proteinmoiety (HSP receptor), phosphatidylserine (scavenger receptors), andlipopolysaccharide (LPS) are additional receptor targets on cells ortissue.

Lectins that can be covalently attached to microparticles to render themtarget specific to the mucin and mucosal cell layer include lectinsisolated from Abrus precatroius, Agaricus bisporus, Anguilla anguilla,Arachis hypogaea, Pandeiraea simplicifolia, Bauhinia purpurea, Caraganarobrescens, Cicer arietinum, Codium fragile, Datura stramonium,Dolichos biflorus, Erythrina corallodendron, Erythrina cristagalli,Euonymus europaeus, Glycine max, Helix aspersa, Helix pomatia, Lathyrusodoratus, Lens culinaris, Limulus polyphemus, Lysopersicon esculentum,Maclura pomifera, Momordica charantia, Mycoplasma gallisepticum, Najamocambique, as well as the lectins Concanavalin A, Succinyl-ConcanavalinA, Triticum vulgaris, Ulex europaeus I, II and III, Sambucus nigra,Maackia amurensis, Limax fluvus, Homarus americanus, Cancer antennarius,and Lotus tetragonolobus.

The attachment of any positively charged ligand, such aspolyethyleneimine or polylysine, to any microparticle may improvebioadhesion due to the electrostatic attraction of the cationic groupscoating the beads to the net negative charge of the mucus. Themucopolysaccharides and mucoproteins of the mucin layer, especially thesialic acid residues, are responsible for the negative charge coating.Any ligand with a high binding affinity for mucin could also becovalently linked to most microparticles with the appropriate chemistry,and be expected to influence the binding of microparticles to the gut.For example, polyclonal antibodies raised against components of mucin orelse intact mucin, when covalently coupled to microparticles, wouldprovide for increased bioadhesion. Similarly, antibodies directedagainst specific cell surface receptors exposed on the lumenal surfaceof the intestinal tract would increase the residence time of beads, whencoupled to microparticles using the appropriate chemistry. The ligandaffinity need not be based only on electrostatic charge, but otheruseful physical parameters such as solubility in mucin or else specificaffinity to carbohydrate groups.

The covalent attachment of any of the natural components of mucin ineither pure or partially purified form to the microparticles woulddecrease the surface tension of the bead-gut interface and increase thesolubility of the bead in the mucin layer. The list of useful ligandswould include but not be limited to the following: sialic acid,neuraminic acid, n-acetyl-neuraminic acid, n-glycolylneuraminic acid,4-acetyl-n-acetylneuraminic acid, diacetyl-n-acetylneuraminic acid,glucuronic acid, iduronic acid, galactose, glucose, mannose, fucose, anyof the partially purified fractions prepared by chemical treatment ofnaturally occurring mucin, e.g., mucoproteins, mucopolysaccharides andmucopolysaccharide-protein complexes, and antibodies immunoreactiveagainst proteins or sugar structure on the mucosal surface.

The attachment of polyamino acids containing extra pendant carboxylicacid side groups, e.g., polyaspartic acid and polyglutamic acid, shouldalso provide a useful means of increasing bioadhesiveness. Usingpolyamino acids in the 15,000 to 50,000 kDa molecular weight range wouldyield chains of 120 to 425 amino acid residues attached to the surfaceof the microparticles. The polyamino chains would increase bioadhesionby means of chain entanglement in mucin strands as well as by increasedcarboxylic charge.

4. Sheddable Polyethylene Glycol (PEG) Coatings

The HPG-coated particles can be modified by covalently attaching PEG tothe surface. This can be achieved by converting the vicinyl diol groupsto aldehydes and then reacting the aldehydes with functional groups onPEG, such as aliphatic amines, aromatic amines, hydrazines and thiols.The linker has end groups such as aliphatic amines, aromatic amines,hydrazines, thiols and O-substituted oxyamines. The bond inserted in thelinker can be disulfide, orthoester and peptides sensitive to proteases.

PEG with a functional group or a linker can form a bond with aldehyde onPLA-HPGALD and reversed the bioadhesive(sticky) state of PLA-HPGALD tostealth state. This bond or the linker is labile to pH change or highconcentration of peptides, proteins and other biomolecules. Afteradministration systematically or locally, the bond attaching the PEG toPLA-HPGALD can be reversed or cleaved to release the PEG in response toenvironment and exposed the PLA-HPGALD particles to the environment.Subsequently, the particles will interact with the tissue and attach theparticles to the tissues or extracellular materials such as proteins.The environment can be acidic environment in tumors, reducingenvironment in tumors, protein rich environment in tissues.

III. Pharmaceutical Compositions

The particles can be formulated with appropriate pharmaceuticallyacceptable carriers into pharmaceutical compositions for administrationto an individual in need thereof. The formulations can be administeredenterally (e.g., oral) or parenterally (e.g., by injection or infusion).Other routes of administration include, but are not limited to,transdermal.

The compounds can be formulated for parenteral administration.“Parenteral administration”, as used herein, means administration by anymethod other than through the digestive tract or non-invasive topical orregional routes. For example, parenteral administration may includeadministration to a patient intravenously, intradermally,intraarterially, intraperitoneally, intralesionally, intracranially,intraarticularly, intraprostatically, intrapleurally, intratracheally,intravitreally, intratumorally, intramuscularly, subcutaneously,subconjunctivally, intravesicularly, intrapericardially,intraumbilically, by injection, and by infusion.

Parenteral formulations can be prepared as aqueous compositions usingtechniques is known in the art. Typically, such compositions can beprepared as injectable formulations, for example, solutions orsuspensions; solid forms suitable for using to prepare solutions orsuspensions upon the addition of a reconstitution medium prior toinjection; emulsions, such as water-in-oil (w/o) emulsions, oil-in-water(o/w) emulsions, and microemulsions thereof, liposomes, or emulsomes.

The carrier can be a solvent or dispersion medium containing, forexample, water, ethanol, one or more polyols (e.g., glycerol, propyleneglycol, and liquid polyethylene glycol), oils, such as vegetable oils(e.g., peanut oil, corn oil, sesame oil, etc.), and combinationsthereof. The proper fluidity can be maintained, for example, by the useof a coating, such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and/or by the use ofsurfactants. In many cases, it will be preferable to include isotonicagents, for example, sugars or sodium chloride.

Solutions and dispersions of the active compounds as the free acid orbase or pharmacologically acceptable salts thereof can be prepared inwater or another solvent or dispersing medium suitably mixed with one ormore pharmaceutically acceptable excipients including, but not limitedto, surfactants, dispersants, emulsifiers, pH modifying agents,viscosity modifying agents, and combination thereof.

Suitable surfactants may be anionic, cationic, amphoteric or nonionicsurface active agents. Suitable anionic surfactants include, but are notlimited to, those containing carboxylate, sulfonate and sulfate ions.Examples of anionic surfactants include sodium, potassium, ammonium oflong chain alkyl sulfonates and alkyl aryl sulfonates such as sodiumdodecylbenzene sulfonate; dialkyl sodium sulfosuccinates, such as sodiumdodecylbenzene sulfonate; dialkyl sodium sulfosuccinates, such as sodiumbis-(2-ethylthioxyl)-sulfosuccinate; and alkyl sulfates such as sodiumlauryl sulfate. Cationic surfactants include, but are not limited to,quaternary ammonium compounds such as benzalkonium chloride,benzethonium chloride, cetrimonium bromide, stearyl dimethylbenzylammonium chloride, polyoxyethylene and coconut amine Examples ofnonionic surfactants include ethylene glycol monostearate, propyleneglycol myristate, glyceryl monostearate, glyceryl stearate,polyglyceryl-4-oleate, sorbitan acylate, sucrose acylate, PEG-150laurate, PEG-400 monolaurate, polyoxyethylene monolaurate, polysorbates,polyoxyethylene octylphenylether, PEG-1000 cetyl ether, polyoxyethylenetridecyl ether, polypropylene glycol butyl ether, Poloxamer® 401,stearoyl monoisopropanolamide, and polyoxyethylene hydrogenated tallowamide. Examples of amphoteric surfactants include sodiumN-dodecyl-.beta.-alanine, sodium N-lauryl-.beta.-iminodipropionate,myristoamphoacetate, lauryl betaine and lauryl sulfobetaine.

The formulation can contain a preservative to prevent the growth ofmicroorganisms. Suitable preservatives include, but are not limited to,parabens, chlorobutanol, phenol, sorbic acid, and thimerosal. Theformulation may also contain an antioxidant to prevent degradation ofthe active agent(s).

The formulation is typically buffered to a pH of 3-8 for parenteraladministration upon reconstitution. Suitable buffers include, but arenot limited to, phosphate buffers, acetate buffers, and citrate buffers.

Water soluble polymers are often used in formulations for parenteraladministration. Suitable water-soluble polymers include, but are notlimited to, polyvinylpyrrolidone, dextran, carboxymethylcellulose, andpolyethylene glycol.

Sterile injectable solutions can be prepared by incorporating the activecompounds in the required amount in the appropriate solvent ordispersion medium with one or more of the excipients listed above, asrequired, followed by filtered sterilization. Generally, dispersions areprepared by incorporating the various sterilized active ingredients intoa sterile vehicle which contains the basic dispersion medium and therequired other ingredients from those listed above. In the case ofsterile powders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof. The powders can be prepared in such a manner that theparticles are porous in nature, which can increase dissolution of theparticles. Methods for making porous particles are well known in theart.

Enteral formulations are prepared using pharmaceutically acceptablecarriers. As generally used herein “carrier” includes, but is notlimited to, diluents, preservatives, binders, lubricants,disintegrators, swelling agents, fillers, stabilizers, and combinationsthereof. Polymers used in the dosage form include hydrophobic orhydrophilic polymers and pH dependent or independent polymers. Preferredhydrophobic and hydrophilic polymers include, but are not limited to,hydroxypropyl methylcellulose, hydroxypropyl cellulose, hydroxyethylcellulose, carboxy methylcellulose, polyethylene glycol, ethylcellulose,microcrystalline cellulose, polyvinyl pyrrolidone, polyvinyl alcohol,polyvinyl acetate, and ion exchange resins.

Carrier also includes all components of the coating composition whichmay include plasticizers, pigments, colorants, stabilizing agents, andglidants. Formulations can be prepared using one or morepharmaceutically acceptable excipients, including diluents,preservatives, binders, lubricants, disintegrators, swelling agents,fillers, stabilizers, and combinations thereof.

Controlled release dosage formulations can be prepared as described instandard references such as “Pharmaceutical dosage form tablets”, eds.Liberman et. al. (New York, Marcel Dekker, Inc., 1989), “Remington—Thescience and practice of pharmacy”, 20th ed., Lippincott Williams &Wilkins, Baltimore, Md., 2000, and “Pharmaceutical dosage Bonus and drugdelivery systems”, 6th Edition, Ansel et al., (Media, Pa.: Williams andWilkins, 1995). These references provide information on excipients,materials, equipment and process for preparing tablets and capsules anddelayed release dosage forms of tablets, capsules, and granules. Thesereferences provide information on carriers, materials, equipment andprocess for preparing tablets and capsules and delayed release dosageforms of tablets, capsules, and granules.

Stabilizers are used to inhibit or retard drug decomposition reactionswhich include, by way of example, oxidative reactions. Suitablestabilizers include, but are not limited to, antioxidants, butylatedhydroxytoluene (BHT); ascorbic acid, its salts and esters; Vitamin E,tocopherol and its salts; sulfites such as sodium metabisulphite;cysteine and its derivatives; citric acid; propyl gallate, and butylatedhydroxyanisole (BHA).

IV. Methods of Making Particles

A. Hyperbranched polyglycerol (HPG)

Hyperbranched polyglycerol can be prepared using techniques known in theart. For example, an initiator having multiple reactive sites is reactedwith glycidol in the presence of a base to form hyperbranchedpolyglycerol (HPG). Suitable initiators include, but are not limited to,polyols, e.g., triols, tetraols, pentaols, or greater and polyamines,e.g., triamines, tetraamines, pentaamines, etc. In one embodiment, theinitiator is 1,1,1-trihydroxymethyl propane (THP).

B. Polymer-HPG Conjugates

Hyperbranched polyglycerol (HPG) can be covalently bound to one or morematerials, such as a polymer, that form the core of the particles usingmethodologies known in the art. For example, HPG can be covalentlycoupled to a polymer having carboxylic acid groups, such as PLA, PGA, orPLGA using DIC/DMAP.

The HPG can be functionalized to introduce one or more reactivefunctional groups that alter the surface properties of the particles.For example, HPG-coated particles prevent non-specific adsorption ofserum proteins and increase the blood circulation of the particles. Suchparticles are referred to as stealth particle. However, the hydroxylgroups on HPG can be chemically modified to cause the particles to stickto biological material, such as tissues, organs, cells, etc. Suchfunctional groups include aldehydes, amines, O-substituted oximes, andcombinations thereof. A synthetic scheme for such chemical conversionsis shown in FIG. 1.

C. Particles

Methods of making polymeric particles are known in the art. Commonmicroencapsulation techniques include, but are not limited to, spraydrying, interfacial polymerization, hot melt encapsulation, phaseseparation encapsulation (spontaneous emulsion microencapsulation,solvent evaporation microencapsulation, and solvent removalmicroencapsulation), coacervation, low temperature microsphereformation, and phase inversion nanoencapsulation (PIN). A brief summaryof these methods is presented below.

In some embodiments, the particles are prepared using an emulsion-basedtechnique. In particular embodiments, the particles are prepared using adouble emulsion solvent evaporation technique. For example, theamphiphilic material and the hydrophobic cationic material are dissolvedin a suitable organic solvent, such as methylene chloride ordichloromethane (DCM), with or without a therapeutic agent. The siRNA isreconstituted in purified water, such as HyPure™ molecular biology gradewater (Hyclone Laboratories, Inc., Logan, Utah). The siRNA solution isadded dropwise to the solution of the amphiphilic material and thehydrophobic cationic material and emulsified to form a first emulsion.The emulsion is added to an aqueous solution of surfactant, such as PVA,to form a double emulsion. The final emulsion is added to water andstirred for an extended period of time (e.g., 3 hours) to allow theorganic solvent to evaporate and the particles to harden. Residualorganic solvent and/or unencapsulated molecules are removed by washing.Other emulsion emulsion-based procedures are described below.

1. Phase Separation Microencapsulation

In phase separation microencapsulation techniques, a polymer solution isstirred, optionally in the presence of one or more active agents to beencapsulated. While continuing to uniformly suspend the material throughstirring, a nonsolvent for the polymer is slowly added to the solutionto decrease the polymer's solubility. Depending on the solubility of thepolymer in the solvent and nonsolvent, the polymer either precipitatesor phase separates into a polymer rich and a polymer poor phase. Underproper conditions, the polymer in the polymer rich phase will migrate tothe interface with the continuous phase, encapsulating the activeagent(s) in a droplet with an outer polymer shell.

2. Spontaneous Emulsion Microencapsulation

Spontaneous emulsification involves solidifying emulsified liquidpolymer droplets formed above by changing temperature, evaporatingsolvent, or adding chemical cross-linking agents. The physical andchemical properties of the encapsulant, as well as the properties of theone or more active agents optionally incorporated into the nascentparticles, dictates suitable methods of encapsulation. Factors such ashydrophobicity, molecular weight, chemical stability, and thermalstability affect encapsulation.

3. Solvent Evaporation Microencapsulation

Methods for forming microspheres using solvent evaporation techniquesare described in E. Mathiowitz et al., J. Scanning Microscopy, 4:329(1990); L. R. Beck et al., Fertil. Steril., 31:545 (1979); L. R. Beck etal, Am. J Obstet. Gynecol., 135(3) (1979); S. Benita et al., J. Pharm.Sci., 73:1721 (1984); and U.S. Pat. No. 3,960,757 to Morishita et al.The polymer is dissolved in a volatile organic solvent, such asmethylene chloride. One or more active agents to be incorporated areoptionally added to the solution, and the mixture is suspended in anaqueous solution that contains a surface active agent such as poly(vinylalcohol). The resulting emulsion is stirred until most of the organicsolvent evaporated, leaving solid microparticles/nanoparticles. Thismethod is useful for relatively stable polymers like polyesters andpolystyrene.

4. Phase Inversion Nanoencapsulation (PIN)

Nanoparticles can also be formed using the phase inversionnanoencapsulation (PIN) method, wherein a polymer is dissolved in a“good” solvent, fine particles of a substance to be incorporated, suchas a drug, are mixed or dissolved in the polymer solution, and themixture is poured into a strong non solvent for the polymer, tospontaneously produce, under favorable conditions, polymericmicrospheres, wherein the polymer is either coated with the particles orthe particles are dispersed in the polymer. See, e.g., U.S. Pat. No.6,143,211 to Mathiowitz, et al. The method can be used to producemonodisperse populations of nanoparticles and microparticles in a widerange of sizes, including, for example, about 100 nanometers to about 10microns.

5. Microfluidics

Nanoparticles can be prepared using microfluidic devices. A polymericmaterial is mixed with a drug or drug combinations in a water miscibleorganic solvent. The water miscible organic solvent can be one or moreof the following: acetone, ethanol, methanol, isopropyl alcohol,acetonitrile and Dimethyl sulfoxide (DMSO). The resulting mixturesolution is then added to an aqueous solution to yield nanoparticlesolution. The targeted peptides or fluorophores or drugs may beassociated with the surface of, encapsulated within, surrounded by,and/or distributed throughout the polymeric matrix of the particles.

Particle Properties

The particles may have any zeta potential. The particles can have a zetapotential from −300 mV to +300 mV, −100 mV to +100 mV, from −50 mV to+50 mV, from −40 mV to +40 mV, from −30 mV to +30 mV, from −20 mV to +20mV, from −10 mV to +10 mV, or from −5 mV to +5 mV. The particles canhave a negative or positive zeta potential. In some embodiments theparticles have a substantially neutral zeta potential, i.e. the zetapotential is approximately 0 mV. In preferred embodiments the particleshave a zeta potential of approximately −30 to about 30 mV, preferablyfrom about −20 to about 20 mV, more preferably from about −10 to about10 mV.

The particles may have any diameter. The particles can have a diameterof between about 1 nm and about 1000 microns, about 1 nm and about 100microns, about 1 nm and about 10 microns, about 1 nm and about 1000 nm,about 1 nm and about 500 nm, about 1 nm and about 250 nm, or about 1 nmand about 100 nm. In preferred embodiments, the particle is ananoparticle having a diameter from about 25 nm to about 250 nm. In morepreferred embodiments, the particles are nanoparticles having a diameterfrom about 180 nm to about 250 nm, preferably from about 180 nm to about230 nm. Particles size typically is based on a population, wherein 60,70, 80, 85, 90, or 95% of the population has the desired size range.

The polydispersity is from about 0.05 to 0.30, preferably from about0.05 to about 0.25, more preferably from about 0.05 to about 0.20, morepreferably from about 0.05 to about 0.15, most preferably from about0.05 to about 0.10.

V. Methods of Using Particles

The particles described herein can be used for a variety of applicationsincluding drug delivery, tissue engineering, etc. In some embodiments,the particles are “stealth” particles, where the hydroxyl groups on HPGincrease circulation in the blood stream by resisting non-specificserum-protein absorption and subsequent uptake by the MPS. This canallow targeted particles to be delivered to the desired site for drugrelease. Alternatively, the vicinyl diol groups can be converted tofunctional groups that adhere to biological materials, such as tissue,organs, cells, proteins, etc. Such particles are referred to as“sticky”.

A. Drug Delivery

PEG has been widely used as a coating in biomaterials and drug deliverysystems. It is commonly accepted that the properties of PEG result froma combination of its neutral charge, molecular flexibility, andhydrophilicity. The use of PEG has become so dominant in the field ofparticulate drug delivery, that alternatives are rarely examined.

HPG is a hyperbranched, hydrophilic polymer with a high density ofhydroxyl groups on its surface: it is more hydrophilic than PEG and hasbeen demonstrated to have better compatibility and non-specificresistance to biomolecules than PEG in certain applications. HPG is wellknown to have a lower intrinsic viscosity than linear PEG, whichdecreases the possibility of red cell aggregation when present in thecirculation.

While HPG has been explored in a variety of biomedical settings,principally for coatings on implanted materials, it has never beforebeen tested as a surface coating for drug delivery systems. The examplesdemonstrate that HPG has substantially improved properties compared toPEG when conjugated to NPs and that these properties result from itshigher hydrophilicity leading to a better effect and more stability insuspension. Therefore, NPs coated with HPG should be more effective inclinical medicine than NPs coated with PEG.

Certain properties of the PLA-HPG conjugate are important for theobserved effects. Because high molecular weight HPG has betterresistance of non-specific adsorption to biomolecules, the low molecularweight components were removed from the synthesized HPG by multiplesolvent precipitations and dialysis.

PLA was selected as the hydrophobic core material because it isbiodegradable, has a long history of clinical use, and is the majorcomponent of a NP system that is advancing in clinical trials. Tocovalently attach the PLA to HPG, the previous approach was to firstfunctionalize the HPG with an amine and then conjugate the carboxylicgroup on PLA to the amine. This approach is efficient but cannot be usedto make HPG as surface coatings since any amines that do not react withPLA will lead to a net positive charge on the neutral HPG surface andreduce the ability of HPG to resist adsorption of other molecules on thesurface. To avoid this, the approach in the examples used a one-stepesterification between PLA and HPG, which maintained the charge neutralstate of the HPG.

Blood circulation and biodistribution are standard methods used toexamine the surface effect in vivo. NPs coated with certain coatingstend to escape the MPS and circulate longer in blood. Although it isknown that PEG coatings can significantly enhance blood circulation ofNPs and reduce accumulation in the liver, the majority of injectedPEG-coated NPs still accumulate in the liver. In contrast, the HPGcoating on PLA NPs produced much lower NP accumulation in the liver.With different markers, labeling and detection methods, the absolutevalue of biodistribution of NPs varies. This makes it difficult tocompare the data to other NP formulations. However the liver to bloodratio of PLA-HPG NPs, approximately ⅓ at 12 hr and approximately 1 at 24hr, is comparable to that of BIND-014, a PLA-PEG NP formulation inclinical trials, that was optimized from more than 100 NP formulations.Surprisingly, the spleen to blood ratio of PLA-HPG NPs, approximately ⅔at 12 h and ˜1 at 24 h, is even lower than that of BIND-014.

Nanoparticles accumulate in tumors through the enhanced permeability andretention (EPR) effect, which results from leaky vasculature of thetumor. Both PLA-HPG NPs and PLA-PEG NPs have a hydrodynamic diameter of100 nm. Notably, tumor accumulation of PLA-HPG NPs is ˜3 times greaterthan accumulation of PLA-PEG NPs. This enhanced tumor accumulation ofPLA-HPG NPs over PLA-PEG NPs may be due to the enhanced bloodcirculation time, which is conferred by their surface coatings.Penetration of the PLA-HPG NPs was further confirmed byimmunohistochemistry.

To demonstrate that PLA-HPG NPs were improved carriers for drugs,therapeutic studies of PLA-HPG NPs having encapsulated therein thechemotherapy drug camptothecin (CPT) were performed on mice bearingsubcutaneous LLC tumors. CPT was selected because it is known to beeffective against a wide variety of tumors, but it is limited inclinical use by very low solubility and side effects. PLA-HPG/CPT NPsprovided significantly better tumor treatment than PLA-PEG/CPT NPs. Inmost respects, the two NPs were similar: PLA-HPG and PLA-PEG NPs hadsimilar weight percentage of surface coating; both PLA-HPG/CPT NPs andPLA-PEG/CPT NPs had enhanced in vitro cytotoxicity; PLA-HPG/CPT NPs weresimilar in size to PLA-PEG/CPT NPs; PLA-HPG/CPT and PLA-PEG/CPT NPsshowed similar in vitro release profiles of CPT. The most notabledifference between the two NP formulations is the presence of HPG versusPEG. Therefore, it is believed that the improved therapeuticeffectiveness of PLA-HPG/CPT NPs is due to the greater emulsionstability, improved blood circulation time, improved biodistribution,and improved tumor penetration that result from HPG.

The submicron size of nanoparticulates offers distinct advantages overlarger systems. First, the small size enables them to extravasatethrough blood vessels and tissue. This is especially important for tumorvessels, which are often dilated and fenestrated with an average poresize less than a micron, compared to normal tissue. Second, solidnanoparticles made from biodegradable polymers and encapsulating drugare ideal for sustained intracellular drug delivery, especially fordrugs whose targets are cytoplasmic. An example of this application withdexamethasone-loaded nanoparticles locally delivered to vascular smoothmuscle cells showed greater and sustained anti-proliferative activitycompared to free drug, indicating more efficient interaction of the drugwith cytoplasmic glucorticoid receptors. The dosage loading variesdepending on the nature of encapsulant. Up to 80% of initial totalamount of agent to be incorporated can be encapsulated in themicroparticles.

The microparticles are useful in drug delivery (as used herein “drug”includes therapeutic, nutritional, diagnostic and prophylactic agents),whether injected intravenously, subcutaneously, or intramuscularly,administered to the nasal or pulmonary system, administered to a mucosalsurface (vaginal, rectal, buccal, sublingual), or encapsulated for oraldelivery. As noted above, the term “microparticle” includes“nanoparticles” unless otherwise stated. The dosage is determined usingstandard techniques based on the drug to be delivered and the method andform of administration. The microparticles may be administered as a drypowder, as an aqueous suspension (in water, saline, buffered saline,etc.), in a hydrogel, organogel, or liposome, in capsules, tablets,troches, or other standard pharmaceutical excipient.

In a preferred embodiment for delivery to a mucosal surface, themicroparticles are modified to include ligands for mucosal proteins orextracellular matrix as described above.

1. Restenosis and Transplantation

Percutaneous transluminal coronary angioplasty (PTCA) is a procedure inwhich a small balloon-tipped catheter is passed down a narrowed coronaryartery and then expanded to re-open the artery. It is currentlyperformed in approximately 250,000-300,000 patients each year. The majoradvantage of this therapy is that patients in which the procedure issuccessful need not undergo the more invasive surgical procedure ofcoronary artery bypass graft. A major difficulty with PTCA is theproblem of post-angioplasty closure of the vessel, both immediatelyafter PTCA (acute reocclusion) and in the long term (restenosis).

The mechanism of acute reocclusion appears to involve several factorsand may result from vascular recoil with resultant closure of the arteryand/or deposition of blood platelets along the damaged length of thenewly opened blood vessel followed by formation of a fibrin/red bloodcell thrombus. Restenosis (chronic reclosure) after angioplasty is amore gradual process than acute reocclusion: 30% of patients withsubtotal lesions and 50% of patients with chronic total lesions will goon to restenosis after angioplasty. Although the exact hormonal andcellular processes promoting restenosis are still being determined, itis currently understood that the process of PTCA, besides opening theartherosclerotically obstructed artery, also injures resident coronaryarterial smooth muscle cells (SMC). In response to this injury, adheringplatelets, infiltrating macrophages, leukocytes, or the smooth musclecells (SMC) themselves release cell derived growth factors withsubsequent proliferation and migration of medial SMC through theinternal elastic lamina to the area of the vessel intima. Furtherproliferation and hyperplasia of intimal SMC and, most significantly,production of large amounts of extracellular matrix over a period of 3-6months, results in the filling in and narrowing of the vascular spacesufficient to significantly obstruct coronary blood flow.

The treatment of restenosis requires additional, generally moreinvasive, procedures, including coronary artery bypass graft (CABG) insevere cases. Consequently, methods for preventing restenosis, ortreating incipient forms, are being aggressively pursued. One possiblemethod for preventing restenosis is the administration ofanti-inflammatory compounds that block local invasion/activation ofmonocytes thus preventing the secretion of growth factors that maytrigger SMC proliferation and migration. Other potentiallyanti-restenotic compounds include antiproliferative agents that caninhibit SMC proliferation, such as rapamycin and paclitaxel. Rapamycinis generally considered an immunosuppressant best known as an organtransplant rejection inhibitor. However, rapamycin is also used to treatsevere yeast infections and certain forms of cancer. Paclitaxel, knownby its trade name Taxol®, is used to treat a variety of cancers, mostnotably breast cancer.

However, anti-inflammatory and antiproliferative compounds can be toxicwhen administered systemically in anti-restenotic-effective amounts.Furthermore, the exact cellular functions that must be inhibited and theduration of inhibition needed to achieve prolonged vascular patency(greater than six months) are not presently known. Moreover, it isbelieved that each drug may require its own treatment duration anddelivery rate. Therefore, in situ, or site-specific drug delivery usinganti-restenotic coated stents has become the focus of intense clinicalinvestigation. Recent human clinical studies on stent-based delivery ofrapamycin and paclitaxel have demonstrated excellent short-termanti-restenotic effectiveness. Stents, however, have drawbacks due tothe very high mechanical stresses, the need for an elaborate procedurefor stent placement, and manufacturing concerns associated withexpansion and contraction.

One of the most promising applications for targeted drug delivery usingnanoparticles is in local application using interventional proceduressuch as catheters. Potential applications have focused on intra-arterialdrug delivery to localize therapeutic agents in the arterial wall toinhibit restenosis (Labhasetwar, et al. J Pharm Sci 87, 1229-1234(1998); Song, et al. J Control Release 54, 201-211 (1998)). Restenosisis the re-obstruction of an artery following interventional proceduressuch as balloon angioplasty or stenting as described above. Drug loadednanoparticles are delivered to the arterial lumen via catheters andretained by virtue of their size, or they may be actively targeted tothe arterial wall by non-specific interactions such as charged particlesor particles that target the extracellular matrix. Surface-modifiednanoparticles, engineered to display an overall positive chargefacilitated adhesion to the negatively charged arterial wall and showeda 7 to 10-fold greater arterial localized drug levels compared to theunmodified nano-particles in different models. This was demonstrated tohave efficacy in preventing coronary artery restenosis in dogs and pigs(Labhasetwar, et al. J Pharm Sci 87, 1229-1234 (1998)). Nanoparticlesloaded with dexamethasone and passively retained in arteries showedreduction in neointimal formation after vascular injury (Guzman, et al.Circulation 94, 1441-1448 (1996)).

The microparticles (and/or nanoparticles) can be used in theseprocedures to prevent or reduce restenosis. Microparticles can bedelivered at the time of bypass surgery, transplant surgery orangioplasty to prevent or minimize restenosis. The microparticles can beadministered directly to the endothelial surface as a powder orsuspension, during or after the angioplasty, or coated onto or as acomponent of a stent which is applied at the time of treatment. Themicroparticles can also be administered in conjunction with coronaryartery bypass surgery. In this application, particles are prepared withappropriate agents such as anti-inflammatories or anti-proliferatives.These particles are made to adhere to the outside of the vessel graft byaddition of adhesive ligands as described above. A similar approach canbe used to add anti-inflammatory or immunosuppressant loaded particlesto any transplanted organs or tissues.

In this embodiment, the drug to be delivered is preferably ananti-proliferative such as taxol, rapamycin, sirulimus, or otherantibiotic inhibiting proliferation of smooth muscle cells, alone or incombination with an anti-inflammatory, such as the steroidalanti-inflammatory dexamethasone. The drug is encapsulated within andoptionally also bound to the microparticles. The preferred size of themicroparticles is less than one micron, more preferably approximately100 nm in diameter. The polymer is preferably a polymer such aspoly(lactic acid-co-glycolic acid) or polyhydroxyalkanoate whichdegrades over a period of weeks to months. Preferably the microparticleshave a high density of an adhesive molecule on the surface such as onethat adds charge for electrostatic adhesion, or one that binds toextracellular matrix or cellular material, or otherwise inert moleculessuch as an antibody to extracellular matrix component. Biotinylatedparticles have a higher level of adhesion to the tissue.

2. Treatment of Tumors

Passive delivery may also be targeted to tumors. Aggressive tumorsinherently develop leaky vasculature with 100 to 800 nm pores due torapid formation of vessels that must serve the fast-growing tumor. Thisdefect in vasculature coupled with poor lymphatic drainage serves toenhance the permeation and retention of nanoparticles within the tumorregion. This is often called the EPR effect. This phenomenon is a formof ‘passive targeting’. The basis for increased tumor specificity is thedifferential accumulation of drug-loaded nanoparticles in tumor tissueversus normal cells, which results from particle size rather thanbinding. Normal tissues contain capillaries with tight junctions thatare less permeable to nanosized particles. Passive targeting cantherefore result in increases in drug concentrations in solid tumors ofseveral-fold relative to those obtained with free drugs.

Passive delivery may also be directed to lymphoid organs of themammalian immune system, such as lymphatic vessels and spleen. Theseorgans are finely structured and specialized in eliminating invadersthat have gained entry to tissue fluids. Nanoparticles may easilypenetrate into lymphatic vessels taking advantage of the thin walls andfenestrated architecture of lymphatic microvessels. Passive targeting tothe spleen is via a process of filtration. Indeed the spleen filters theblood of foreign particles larger than 200 nm. This function facilitatessplenic targeting with nanoparticles encapsulating drug for effectivetreatments against several hematological diseases.

Both liposomal and solid nanoparticles formulations have receivedclinical approval for delivery of anticancer drugs. Liposomalformulations include those of doxorubicin (DOXIL®1/CAELYX®1 ANDMYOCET®1) and daunorubicin (DAUNOSOME®1). The mechanism of drug releasefrom liposomes is not clear, but is thought to depend on diffusion ofthe drug from the carrier into the tumor interstitium. This is followedby subsequent uptake of the released drug by tumor cells. The mechanismof release is still poorly understood, which hinders advancedapplications involving the addition of active ligands for cellulartargeting in vivo. Recently, the FDA approved ABRAXANE®, analbumin-bound paclitaxel nanoparticles formulation as an injectablesuspension for the treatment of metastatic breast cancer. In addition,other solid nanoparticle-based cancer therapies have been approved forclinical trials, for example a Phase 1 clinical trial has been approvedthat will evaluate the safety of hepatic arterial infusion of REXIN-G™(a targeted nanoparticle vector system with a proprietary mutantcell-cycle control gene, i.e. anti-cancer gene) as an intervention forcolorectal cancer.

The particles described herein should be efficacious in the treatment oftumors, especially those where targeting is beneficial and delivery ofhigh doses of chemotherapeutic desirable. An important feature oftargeted particle delivery is the ability to simultaneously carry a highdensity of drug while displaying ligands on the surface of the particle.It is well known that other drug carrier systems, such as immunotoxinsor drug-immunoconjugate, which are made by tethering drug molecules toantibodies or synthetic polymers, usually deliver less than 10 drugmolecules per carrier to target cells. Targeted high densitynanoparticles on the other hand can deliver thousands of drug moleculeson the surface, and millions of molecules in their interior.

One important target is E-selectin, which is involved in the arrest ofcirculating immune system cells and is differentially upregulated withinflammatory and immune processes and should be useful to enhancedelivery of therapeutic agents to the vasculature including tumor bloodvessels through selective targeting. A second important class of targetsis receptors involved in the uptake of vitamin B12, folic acid, biotinand thiamine. These are differentially overexpressed on the surface ofcancer cells creating a possible target for several types of cancer,including ovarian, breast, lung, renal and colorectal cancers. One ofthe most promising strategies for enhancing active immunotherapy andinducing potent vaccination is targeting of antigen-loaded nanoparticlesto antigen-presenting cells such as dendritic cells (DCs). Nanoparticlesincorporating toll-like receptors (TLRs) in biodegradable PLGA haveshown efficient delivery of antigen to DC and potent activation of the Tcell immune response.

The overall strength of nanoparticles binding to a target is a functionof both affinity of the ligand-target interaction and the number oftargeting ligands presented on the particle surface. Nanoparticlesproduced by the present techniques have many thousands of ligands ontheir surface. This is a particularly useful feature for ligands that intheir monomer form have a weak affinity to their target receptors, suchas single chain variable fragments (scFv), which in most cases must bereengineered into multimers to increase their avidity of interaction totarget cells or peptide/Major histocompatability complex (peptide/MHC),which have weak affinity to target T cell receptors. For example,multivalency increases the avidity of interaction of peptide/MHC to theT cell up to 100 fold facilitating enhanced interactions and effectivedrug delivery to target antigen-specific T cells.

3. Macular Degeneration

Macular degeneration (MD) is a chronic eye disease that occurs whentissue in the macula, the part of the retina that is responsible forcentral vision, deteriorates. Degeneration of the macula causes blurredcentral vision or a blind spot in the center of your visual field.Macular degeneration occurs most often in people over 60 years old, inwhich case it is called Age-Related Macular Degeneration (ARMD) or(AMD). AMD is the leading cause of blindness in the United States andmany European countries. About 85-90% of AMD cases are the dry,atrophic, or nonexudative form, in which yellowish spots of fattydeposits called drusen appear on the macula. The remaining AMD cases arethe wet form, so called because of leakage into the retina from newlyforming blood vessels in the choroid, a part of the eye behind theretina. Normally, blood vessels in the choroid bring nutrients to andcarry waste products away from the retina. Sometimes the fine bloodvessels in the choroid underlying the macula begin to proliferate, aprocess called choroidal neovascularization (CNV). When those bloodvessels proliferate, they leak, causing damage to cells in the maculaoften leading to the death of such cells. The neovascular “wet” form ofAMD is responsible for most (90%) of the severe loss of vision. There isno cure available for “wet” or “dry” AMD.

The exact causes of AMD are not known, however, contributing factorshave been identified. Factors that contribute to AMD include reactiveoxidants which cause oxidative damage to the cells of the retina and themacula, high serum low density cholesterol lipoprotein (LDL)concentration, and neovascularization of the choroid tissue underlyingthe photoreceptor cells in the macula.

Treatments for wet AMD include photocoagulation therapy, photodynamictherapy, and transpupillary thermotherapy. AMD treatment withtranspupillary thermotherapy (TTT) photocoagulation is a method ofdelivering heat to the back of the patient's eye using an 810 nminfrared laser, which results in closure of choroidal vessels. AMDtreatment with photocoagulation therapy involves a laser aimed atleakage points of neovascularizations behind the retina to preventleakage of the blood vessel. Photodynamic therapy (PDT) employs thephotoreactivity of a molecule of the porphyrin type, called verteporphinor Visudyne, which can be performed on leaky subfoveal or juxtafovealneovascularizations. MACUGEN® is an FDA approved drug that inhibitsabnormal blood vessel growth by attacking a protein that causes abnormalblood vessel growth.

Other potential treatments for “wet” AMD that are under investigationinclude angiogenesis inhibitors, such as anti-VEGF antibody, andanti-VEGF aptamer (NX-1838), integrin antagonists to inhibitangiogenesis has also been proposed, and PKC412, an inhibitor of proteinkinase C. Cytochalasin E (Cyto E), a natural product of a fungal speciesthat inhibits the growth of new blood vessels is also being investigatedto determine if it will block growth of abnormal blood vessels inhumans. The role of hormone replacement therapy is being investigatedfor treatment of AMD in women.

There are no treatments available to reverse “dry” AMD. Treatments shownto inhibit progression of AMD include supplements containingantioxidants. The use of a gentle “sub-threshold” diode laser treatmentthat minimizes damage to the retina is being investigated for treatmentof “dry” AMD. Another potential treatment for AMD includes rheopheresis,which is a form of therapeutic blood filtration that removes “vascularrisk factor” including LDL cholesterol, fibrinogen, and lipoprotein A.Rheopheresis has not yet been FDA-approved, but is available in Canadaand Europe. Other treatments for AMD under investigation includeculturing and transplantation of cells of the Retinal Pigment Epithelium(RPE), metalloproteinase modulators, inhibitors of A2E, a vitamin Aderivative, which accumulates in the human eye with age, andcarotenoids, zeaxanthin and lutein.

There have been a number of studies indicating that macular degenerationis caused by, or associated with, a defect in complement factor H(Haines, et al. Science. 2005 308(5720):419-21; Edwards, et al. Science.2005 15; 308(5720):421-4; Klein, et al. Science. 2005; 308(5720):385-9).This leads to a method of treatment or prevention of the maculardegeneration through administration of one of the known complementinhibitors, such as antibodies (antibody fragments, recombinantantibodies, single chain antibodies, humanized and chimeric antibodies)to C3b or a component thereof. An example is PEXELIZUMAB® (AlexionPharmaceuticals, Inc., Cheshire, Conn., USA), a humanized, monoclonal,single-chain antibody fragment that inhibits C5, thereby blocking itscleavage into active forms. A potential inhibitor is relatively small,broad-acting C inhibitory protein (termed OmCI), described by Nunn, etal. J Immunol. 2005 15; 174(4):2084-91.

Ocular delivery of drug-loaded, sustained-release and optionallytargeted nanoparticles by intravitreal administration is a promisingroute for eye disease because it eliminates the need for multipleinjections of drug into the eye. Coupled with the problem of retentionof adequate concentrations of therapeutic agent in the pre-corneal area(Mainardes, et al. Curr. Drug Targets 6, 363-371 (2005)), biodegradablenanoparticles delivered intravitreally have demonstrated localization inthe retinal pigment epithelium (Bourges, et al. Invest. Ophthalmol. VisSci 44, 3562-3569 (2003)) and greater therapeutic efficacy in oculardisease such as autoimmune uveoretinitis (de Kozak, et al. Eur. J.Immunol. 34, 3702-3712 (2004)).

In this embodiment, the drug is encapsulated with, and optionally alsobound to the microparticles. The preferred size of the microparticles isapproximately 100 nm in diameter. The polymer is preferably a polymersuch as poly(lactic acid-co-glycolic acid) or polyhydroxyalkanoate whichdegrades over a period of weeks to months.

In the preferred embodiment, degradable particles less than one micronin diameter, preferably about 100 nm in diameter, are distributed withinthe eye by subretinal injection or intravitreally injection, where theydegrade over a period of from several weeks to several months. In themost preferred case, the microparticles have a high density of adhesivemolecules to retinal epithelial cells.

B. Tissue Engineering Matrices and Wound Healing Dressings

The microparticles can be dispersed on or within a tissue engineeringmatrix for delivery of growth factors or modulatory compounds, asdemonstrated in the examples. Many types of materials are known for usein tissue engineering, including materials formed of synthetic polymer,decellularized matrix, collagen, and decellularized tissue. These can bein the form of fibrous matrices or materials such as those used in bonerepair or replacement, which consist primarily of materials such ashydroxyapatite. In another embodiment, nanoparticles deliveringmolecules which are used to enhance wound healing such as antibiotics,growth, angiogenesis stimulating molecules, and other types of drugs,can be applied to wound healing matrices, implants, dressings, bonecements, and other devices which are applied to the site of injury.Preferred antibiotics include vancomycin, ciprofloxacin andanti-infective peptides such as the defensin molecules. In addition,re-vascularization of these grafts can be a problem, hence VEGF, FGF andPDGF could be included in the particles.

The advantage of these particles is that they adhere to theimplanted/applied material, where they are retained at the site ofinjury to provide sustained treatment. Mixtures releasing differentamounts or different drugs at different times are particularlyadvantageous for treatment of wounds such as diabetic wound ulcers.Ligands can be selected to enhance the particles being retained at thesite, by binding to extracellular matrix or through non-specificelectrostatic binding. In addition, other ligands can be selected toenhance the interaction of particles or matrix with cells that areeither added to the material prior to implantation or migrate into thematerial after implantation.

EXAMPLES

Materials and Methods

Polylactic acid (Mw=20.2 kDa, Mn=12.4 kDa) was obtained from Lactel.

H₂N-PEG(5000)-OCH₃ was obtained from Laysan.

Anhydrous dimethylformide, dichloromethane, diisopropylcarboimide,dimethylaminopyridie, potassium methoxide, camptothecin, polyvinylalcohol, paraformaldehyde, TWEEN® 80, and 1,1,1-trihydroxymethyl propanewere obtained from the Sigma-Aldrich.

Anhydrous dry ether, methanol, acetonitrile and dimethylsulfoxide wereobtained from J. T. Baker.

1,1′-Dioctadecyl-3,3,3′,3′-Tetramethylindodicarbocyanine,4Chlorobenzenesulfonate Salt (DiD) and DAPI stain were obtained fromInvitrogen.

Super frost microscope slides were obtained from Thermo Scientific.

Donkey normal serum and Rabbit-anti-CD31 antibody were obtained fromAbeam and the Donkey-anti-rabbit secondary antibody tagged with Alexa488fluorophore was obtained from Invitrogen.

Cell titer blue was obtained from Promega.

Microdialysis tubing was from Thermo Scientific.

Phorbol 12-myristate 13-acetate (PMA) was from Abcam.

Cell Lines

Lewis lung carcinoma (LLC) cell line was obtained from the American TypeCulture Collection (ATCC) (Manassa, Va.). LLC cells were maintained inDulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetalbovine serum (FBS) and 1% penicillin-streptomycin at 37° C. under 5% CO2humidified atmosphere. U937 was maintained in RPMI1640 supplemented with10% FBS. Differentiation of U937 to macrophage was induced by PMA (50ng/ml).

Example 1. Synthesis of Hyperbranched Polyglycerol

Hyperbranched polyglycerol (HPG) was synthesized by anionicpolymerization. Briefly, 4.6 mmol 1,1,1-trihydroxypropane (THP) wasadded into an argon protected flask in a 95° C. oil bath and 1.5 mmolKOCH₃ was added. The system was hooked up to a vacuum pump and leftunder vacuum for 30 min. The system was refilled with argon and 25 mlglycidol was added by a syringe pump over 12 hours. The HPG wasdissolved in methanol and precipitated by addition of acetone. HPG waspurified 2-3 times with methanol/acetone precipitation. To furtherremove the low molecular weight HPG, 2-5 ml HPG was placed in a 10 mldialysis tube (0.5-1 k cut-off) and dialyzed against deionized (DI)water. The water was replaced two times every 12 hours. HPG wasprecipitated with acetone and then dried under vacuum at 80° C. for 12h.

Example 2. Synthesis of PLA-HPG and PLA-PEG Copolymers

PLA (5 g) and 2.15 g HPG were dissolved in dimethyl formamide (DMF) anddried over molecular sieves overnight. 0.06 ml diisopropylcarboimide(DIC) and 10 mg 4-(N,N-dimethylamino)pyridine (DMAP) were added and thereaction proceeded for 5 days at room temperature under stirring. Theproduct was precipitated by pouring the reaction into cold diethyl ether(ether) and collecting the precipitate by centrifugation. The productwas redissolved in dichloromethane (DCM) and precipitated again with acold mixture of ether and methanol. The product was washed with a coldmixture of ether and methanol. The polymer was dried under vacuum for 2days.

To synthesize PLA-PEG, 2.6 g PLA and 1.0 g MPEG-NH₂ were dissolved inDMF and dried over molecular sieves overnight. 0.038 ml DIC was addedand the reaction proceeded for 2 days at room temperature understirring. The product was precipitated by pouring the reaction into coldether and collecting the precipitate by centrifugation. The product wasredissolved in DCM and precipitated again with cold ether, washed with acold mixture of ether and methanol and dried under vacuum for 2 days.

Example 3. Fabrication of Nanoparticles (NPs)

Fifty mg of PLA-HPG copolymer dissolved in 1.5-3.0 ml of ethylacetate/dimethyl sulfoxide (DMSO) (4:1) was added to 4 ml DI water undervortexing and subjected to probe sonication for 3 cycles at 10 sec each.The resulting emulsion was diluted in 20 ml DI water under stirring. Itwas stirred for at least 5 hours or attached to a ratovapor to evaporatethe ethyl acetate and then applied to an AMICO® ultra centrifugefiltration unit (100 k cut-off). The NPs were washed by filtration 2times then suspended in a 10% sucrose solution. The NPs were kept frozenat −20° C.

The PLA-PEG NPs were made using a single emulsion technique. 50 mgPLA-PEG copolymer dissolved in 1.5-3.0 ml ethyl acetate/DMSO (4:1) wasadded to 4 ml DI water with 2.5% PVA under vortexing and subjected toprobe sonication for 3 cycles of 10 sec each. The resulting emulsion wasdiluted in 20 ml DI water with 0.1% TWEEN® 80 with stirring. Theemulsion was stirred for at least 5 hours or attached to a ratovapor toevaporate the ethyl acetate and then the solution was applied to anAmico ultra centrifuge filtration unit (100 k cut-off). The NPs werewashed by filtration for 2 times then suspended in a 10% sucrosesolution.

¹H NMR spectra for HPG and PLA-HPG block-copolymer were recorded on a400 MHz Agilent instrument using DMSO-d6 as solvent. Inverse gated ¹³CNMR spectra for HPG were recorded on a 600 MHz Agilent instrument withmethanol-d4 as solvent.

The DP_(n) (number-average degree of polymerization) for HPG wascalculated according to the inverse gated ¹³C NMR spectra for HPG withthe following equation:

$\overset{\_}{{DP}_{n}} = {\frac{\left( {T + L_{13} + L_{14} + D} \right)}{\left( {T - D} \right)}f_{c}}$The functionality of the core molecule (TMP), f_(c), is 3.The Mn of HPG is calculated with the following equation:Mn=Molecular weight of glycidol×DP_(n) of HPG+molecular weight of TMP.

Both particles have a biodegradable PLA core, which can be used to loadhydrophobic agents, and a hydrophilic shell of HPG or PEG. HPG was madeby anionic polymerization and characterized by ¹H NMR and ¹³C NMR.PLA-HPG copolymer was synthesized by esterification and the conjugationof PLA-HPG was confirmed by ¹H NMR. The weight percentage of HPG inPLA-HPG was about 29% as calculated from the NMR results.

PLA-HPG NPs were made from a single emulsion as described above. PLA-PEGcopolymer was synthesized by the conjugation of PLA-COOH with amineterminated mPEG and also characterized with ¹H NMR. The weightpercentage of PEG was about 26% as calculated from the NMR results.

Example 4. Characterization of Nanoparticles (NPs) by TransmissionElectron Microscopy (TEM)

Materials and Methods

The NPs were characterized with TEM. A drop of nanoparticle suspensionwas applied on the top of carbon coated copper grids and most of thedroplet was removed with a piece of filter paper. The thin layer of NPssuspension was dried for 5-10 min and then a droplet of uranyl acetatewas applied. Most of the droplet was removed with a filter paper andleft to dry for 5 min. The sample was mounted for imaging with TEM. Thesize distribution of NPs was analyzed in Image J. The hydrodynamic sizeof NPs was determined by dynamic laser scattering (DLS). NPs suspensionwas diluted with DI water to 0.05 mg/ml and 1 ml was loaded into thecell for detection.

To determine the concentration of the dye in NPs, 990 μL DMSO was addedto 10 μL NPs in aqueous solution. The solution was vortexed and left inthe dark for 10 min. The concentration of the dye was quantified with aplate reader by fluorescence of the DiD dye at 670 nm with an excitationwavelength at 644 nm.

The amount of CPT encapsulated in NPs was determined by fluorescence ofCPT at 428 nm with an excitation wavelength at 370 nm. One volume of NPsuspension was diluted in acidified DMSO (1N HCl:DMSO=1:100, volumeratio) at least 10 fold. The fluorescence of CPT was measured and theamount of CPT was determined by comparing to a standard curve.

Results

Transmission electronic microscopy (TEM) confirmed the spherical shapeof the PLA-HPG and PLA-PEG NPs (FIGS. 2A, 2B, 2C and 2D). Thehydrodynamic diameter of NPs was 100 nm as measured by dynamic lightscattering (DLS) (Table 1). In this study, CPT loading of both PLA-HPGand PLA-PEG NPs is 5%. The NPs loaded with CPT have a larger fraction inthe upper size range and larger size of hydrodynamic diameters by TEMimaging. When the NPs were loaded with CPT and incubated in bufferedwater, the agent was released over a period of about 1 week (FIG. 3).Both NPs showed similar patterns of CPT release. After 24 hr ofincubation, over half of the CPT was released from PLA-HPG (59%) andPLA-PEG (56%) NPs. The rest of the encapsulated drug was slowly releasedover a period of 1 week. PLA-HPG/CPT NPs remain suspended in solutionsignificantly longer than PLA-PEG/CPT NPs, indicating greater stabilityof PLA-HPG/CPT NPs in suspension.

TABLE 1 Average diameter of PLA-HPG nanoparticles, PLA- PEGnanoparticles, PLA-HPG/camptothecin (CPT) nanoparticles, and PLA-PEG/CPTnanoparticles. NPs Diameter (nm) PLA-HPG 102.1 ± 3.1 PLA-PEG 103.3 ± 1.0PLA-HPG/CPT 158.4 ± 8.8 PLA-PEG/CPT 142.3 ± 5.2

Example 5. Evaluation of NPs In Vitro

Materials and Methods

A suspension containing 3 mg NPs in a dialysis tube (10K cut-off) wasdialyzed against 40 ml PBS. At each time point, 970 μL solution wasremoved and the rest was replaced with 40 ml fresh PBS. To quantify theCPT in the 970 μL dialyte, 30 μL of quantification fluid (DMSO:10% SDS:1N HCL=1:1:1, volume ratio) was added and the CPT concentration wasquantified at EX/EM 370/428 nm with a plate reader.

Microdialysis tubes were filled with 100 μL of NPs loaded with1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine,4-chlorobenzenesulfonate Salt (DiD) and placed on a floater in a largebeaker with 4 L PBS at 37° C. Tubes were removed in triplicates atdifferent time points. The PBS was changed every 12 hours. The dye leftin the dialysis tube was quantified by fluorescence.

180 μL of LLC cells was plated in each well of a 96 well plate at adensity of 5,000/well and left in a 37° C. incubator overnight. 20 μl offree CPT or NPs in medium were added to each well. The cells wereincubated for 72 h and cell viability was quantified with Cell TiterBlue.

The surface properties of PLA-HPG NPs were evaluated in vitro bymeasuring cell uptake by macrophages. Both PLA-HPG NPs and PLA-PEG NPsshowed significantly lower cell uptake compared to that of the plain PLANPs. PLA-HPG/CPT NPs were evaluated for cell toxicity to LLC cells. Thecontrols chosen for this study were PLA-PEG/CPT NPs and free CPT.

Results

Both NP formulations showed the significantly improved cytotoxicityprofile (FIG. 4A). To show that this toxicity is due to the CPT, and notthe polymers, we examined the effect of blank NPs on LLC cells: bothblank NP formulations showed no toxicity (FIG. 4B).

Example 6. Evaluation of NPs in Blood Circulation and Biodistribution

Materials and Methods

All animal care and studies were approved by Yale's Institutional AnimalCare and Use Committee (IACUC). Both NPs were loaded with 0.2%fluorescence dye (DiD from Invitrogen). 14 C57BL/6 mice (n=7 per group)received tail vein injection of 150 μL DiD loaded NPs (3 mg/ml in PBSsolution). At 5 min, 15 min, 30 min, 1 hr, 2 hr, 4 hr, 8 hr, 24 hr, 48hr and 72 hr 10-20 μL blood were collected from each mouse by tailsnipping. The blood was lyophilized. To quantify the fluorescence in theblood, 100 μl of DMSO and 1 ml of acetonitrile were added andhomogenized with a homogenizer. The homogenized solution was spun on abenchtop centrifuge at 13,000 RPM and then 0.8 ml supernatant wasremoved and added to an eppendorf tube. All the acetonitrile wasevaporated with a SpeedVac and the dye in the DMSO was quantified atEx/Em 644/670 nm with a plate reader.

The biodistribution of DiD-loaded NPs was evaluated in Balb/C micebearing subcutaneous LLC tumors. LLC cells (1×10⁶ cells, 0.1 ml) wereinjected subcutaneously into Balb/C female mice (6 week old, CharlesRiver Laboratories), and nanoparticle administration was started after 7days, a time when the average tumor volume reached approximately 100mm³.

Thirty mice were divided into four groups with 7-8 animals in eachgroup. The average size and size variation of the tumors in all groupswere comparable. 150 μL DiD loaded NPs (3 mg/ml in PBS solution) wereadministrated intravenously through tail vein. At 12 h and 24 h, themice were euthanized and blood was collected with cardiac puncture.After perfusion through the left ventricle with PBS, the organs werecollected. The blood and organs were lyophilized.

To quantify the dye in the organs and tumors except lung and spleen, 1ml DMSO was added and homogenized. The homogenized solution was spun ona benchtop centrifuge at 13,000 RPM and then 0.1 ml of the supernatantwas added to a 96 well plate and the dye in the DMSO was quantified atEx/Em 644/670 nm with a plate reader.

To quantify the dye in lung and spleen, 1 ml of DMSO was added andhomogenized. The homogenized solution was spun on a benchtop centrifugeat 13,000 RPM and then 0.1 ml supernatant was added to 1 mlacetonitrile. The solution was spun on a benchtop centrifuge at 13,000RPM and then 0.8 ml supernatant was removed and added to an eppendorftube.

All the acetonitrile was evaporated with a SpeedVac and the dye wasquantified in the DMSO at Ex/Ex 644/670 nm with a plate reader. One-wayanalysis of variance (ANOVA) was performed to determine the statisticalsignificance of the dose distribution in organs and blood, p<0.05 wasconsidered to be significant.

To demonstrate the effect of the HPG coating on the nanoparticle surfacein vivo, NPs were injected intravenously and blood analyzed periodicallyfor the presence of particles. To permit quantification of NPconcentration in the blood, NPs were loaded with 0.2%1,1′-Dioctadecyl-3,3,3′,3′-tetramethylindo-dicarbocyanine,4-Chlorobenzenesulfonate Salt (DiD). DiD is a hydrophobic dye, which hasbeen widely used as a marker for NPs.

Results

DiD-loaded NPs release a minimal amount of dye (approximately 20%) over5 days of continuous incubation in PBS (FIG. 5). Both PLA-HPG andPLA-PEG NPs were loaded with equivalent amounts of DiD. Comparing thePLA-PEG NPs, the PLA-HPG showed a longer time of circulation afteradministration (FIG. 6A). The elimination half-life of PLA-HPG NPs (10.3hr) was significantly longer than PLA-PEG NPs (6.8 hr): half-lives weredetermined by fitting with a two-compartment model (FIG. 6B). Anegligible amount of either NP was found in circulation 2 days afteradministration.

To further demonstrate the effect of the PLA-HPG NPs, thebiodistribution of the NPs in mice with subcutaneous LLC tumors wasstudied. Fluorescence measured in tissues was normalized to percentdose/gram tissue (FIGS. 6C and 6D). There was no significant differencein the accumulation of either PLA-HPG or PLA-PEG NPs in brain, heart,kidney, lung and spleen at 12 and 24 hr. However, compared to thePLA-PEG NPs, the PLA-HPG NPs were present in significantly higherconcentration in the tumor and blood, but significantly lowerconcentration in the liver at 12 hr after injection. These differencespersisted at 24 hr (although were not statistically significant in bloodand tumor). To better understand the impact of overall NP distributionwe calculated the total mass of NPs in each organ, tumor and the wholeblood by multiplying the percent dose/gram tissue with tissue weight(FIGS. 6E and 6F). The majority of NPs were in either liver or blood atboth time points. At 12 hours, two times as many PLA-HPG NPs werepresent as PLA-PEG NPs in whole blood and one third as many PLA-HPG NPs,compared to PLA-PEG NPs, were present in the liver. At 24 hours, PLA-HPGaccumulation in the liver was half of PLA-PEG though both NPs werepresent in similar quantities in blood.

Example 7. Immunohistochemistry

Materials and Methods

Nine balb/c mice (n=3 per group) bearing LLC tumors underwent tail veininjection of 150 μL NPs (3 mg/ml in PBS solution) or PBS control. At 12hrs, animals were sacrificed. After perfusion through the left ventriclewith PBS the tumors were dissected and frozen in OCT. The tumors weresectioned at 10 μm thickness and immobilized onto SUPERFROST® Microscopeslides. The tumor sections were fixed in 4% paraformaldehyde in PBS for30 min and then washed with TBS (20 mM Tris PLA-HPG 7.6, 140 mM NaCl) 3times 5 min each. Samples were blocked in TBS with 1% BSA and 5% donkeynormal serum for 1 h and then incubated with Rabbit-anti-CD31 antibody(1:50 dilution in TBS with 1% BSA and 5% donkey normal serum). Thesections were washed with TBS 3 times 5 min each and then incubated withDonkey-anti-rabbit secondary antibody tagged with Alexa488 fluorophore(Invitrogen, 1:200 dilution in TBS with 1% BSA and 5% donkey normalserum) for 1 hour and washed again with TBS 3 times, 5 min each. Severaldrops of DAPI were placed on each slide and the slides were covered witha coverslip. Images were taken with a Zeiss fluorescence microscope.

Results

To visualize the penetration of NPs in tumors, immunohistochemistry oncryo-sections of tumors that were treated with PLA-HPG NPs wasperformed. NPs were found beyond the boundaries of blood vessel lumens,indicating that the NPs penetrated deep into the tumor tissue afterextravasating through the tumor vascular after intravenousadministration.

Example 8. Therapeutic Studies

Materials and Methods

Forty C57BL/6 mice (6 weeks old, Charles River Laboratories) weresubcutaneously injected 1×10⁶ LLC cells. After 7 days, a time when theaverage tumor volume reached approximately 100 mm³, 40 mice were equallydivided into 5 groups with comparable average size distribution oftumors. After grouping the mice, drug treatments were startedimmediately with PBS control, blank PLA-HPG NPs control and 3 CPTformulations: 1) PBS (PLA-HPG=7.4); 2) blank PLA-HPG NPs in PBS; 3) CPTDMSO solution (2.5 mg/ml, DMSO:PBS10×=9:1 volume ratio); 4) PLA-PEG/CPTNPs in PBS; 5) PLA-HPG/CPT NPs in PBS. Treatments were administrated 2times at 7 and 11 days with a dose of CPT (5 mg/kg) each time. The tumorvolumes and body weights of the mice were measured and recorded everytwo days. The tumor volumes were calculated with the formulaVolume=LW2/2, where L and W are the long and short diameter of a tumorrespectively. Animals were euthanized when the tumor size exceeded 2000mm³, total body weight loss exceeded 20%, or when other signals ofsickness, such as breathing problems, failure to eat and drink, lethargyor abnormal posture, were observed. One-way ANOVA analysis was performedto determine the statistical significance of treatment-related changesin tumour volume of animals and p<0.05 was considered to be significant.

Results

To compare the therapeutic effect of PLA-HPG NPs with the optimizedPLA-PEG NPs, CPT-loaded NPs intravenously were injected in mice bearingLLC subcutaneous tumors at a CPT dose of 5 mg/kg at 7 and 11 days aftertumor inoculation (FIG. 7A). The growth rate of tumors treated with theblank PLA-HPG NPs was indistinguishable from that of tumors treated withPBS control, indicating that PLA-HPG alone has no effect on the tumorgrowth, as we anticipated. The tumor growth rate in mice givenPLA-HPG/CPT NPs was significantly lower than that of the mice treatedwith PBS, free CPT, or PLA-PEG/CPT NPs. Interestingly, no significant invivo toxicity was observed for all formulations (FIG. 7B).

Example 9. Synthesis of Functionalized HPG-Coated Nanoparticles andEvaluation of Reversibility of Stealth Properties of Nanoparticles inBlood Circulation

Materials and Methods

Synthesis of Aldehyde Functionalized Nanoparticles PLA-HPG NPs (0.1mg/ml) in a 96-well plate (small vial) were with 2 mM Na₂SO₃. The NPswere washed two times with DI water in an ACROPREP filter plate with 100k cut-off (or AMICON® ultra filter 0.5 ml with 100 k cut-off) and thensuspended in DI water.

The aldehydes on NPs were quantified with an aldehyde quantificationassay kit (ABCAM®). The PLA-HPG NPs were used as a backgroundsubtraction control. The amount of aldehyde was calculated by comparingto a reference curve. The reference curve was made by using the aldehydestandard provided with the kit. The amount of aldehyde on each particlewas calculated based on 100 nm hydrodynamic diameter of NPs and anassumed NP density of 1.0 g/cm³. For microarray printing, NPs load withDiD dye were suspended in PBS buffer containing 15% glycerol and 0.01%TRITON®-X100 at a concentration of 1 mg/ml in a 384-well plate. The NPswere arrayed on lysine coated slides using a SPOTBOT® microrrayer fromARRAYIT®. After 1 hour incubation in a humidity chamber, the printedslides were washed extensively with PBS 3 times, 5 min each. After aquick rinse with DI water, the slides were blow-dried with argon andsubjected for imaging.

Ligand Attachment

For ligand or protein attachment, in a 96-well plate (or small vials),PLA-HPG_(ALD) NPs were incubated with ligands or proteins (NaCNBH₄should be added for proteins or ligands modified with amines orhydrazines) for 1 min-12 hours and the reaction was quenched with anexcess amount of hydroxylamine (or ethanolamine for proteins or ligandsmodified with amines or hydrazines) solution in TRIS buffer (PH=7.4).The NPs were transferred to an AcroPrep filter plate with 100 k cut-off(or amicon ultra filter 0.5 ml with 100 k cut-off or gel filtration forproteins and other large molecules) and washed two times with DI wateror buffer.

To reduce the PLA-HPG_(ALD) NPs back to PLA-HPG NPs (also referred toherein as non-bioadhesive nanoparticles, NNPs), PLA-HPG_(ALD) NPs wereincubated with NaBH₄ in NaH₂PO₄ (0.2M, PH=8.0) and the reaction wasquenched with acetic acid and neutralized with PBS buffer. The NPs werewashed with DI water twice. The blood circulation experiments wereperformed using the method in Example 6.

Polylysine coated glass slides were used as a tissue mimic to evaluatethe bioadhesive property of PLA-HPG_(ALD) NPs (BNPs). PLA-HPG_(ALD) NPswith different concentrations of aldehydes were prepared using ahigh-throughput procedure, where regular 96-well plates and 96-wellfilter plates were used to prepare the NPs and printed onto polylysinecoated slides with a microarrayer.

For microarray printing, NPs load with DiD dye were suspended in PBSbuffer containing 15% glycerol and 0.01% TRITON®-X100 at a concentrationof 1 mg/ml in a 384-well plate. The NPs were arrayed on lysine coatedslides using a SPOTBOT® microrrayer from ARRAYIT®. After 1 hourincubation in a humidity chamber, the printed slides were washedextensively with PBS 3 times, 5 min each. After a quick rinse with DIwater, the slides were blow-dried with argon and subjected for imaging.

The bioadhesive property of PLA-HPG NPs on tissues was evaluated byapplying suspended NPs ex vivo to the luminal surface of human umbilicalvein. The umbilical cord was obtained from the Vascular Biology &Therapeutics Core Facility at Yale University and used within 12 hours.

The umbilical cord was cut into 10 cm length and washed with Ringer'sbuffer. The vein was perfused with 30 ml Ringer's buffer. PLA-HPG NPsand PLA-HPG_(ALD) NPs (1 mg/ml) in Ringer's buffer were injected intovein and both ends of the vein were sealed. The sealed umbilical cordswere immersed into Ringer's buffer and incubated at 37° C. for 2 h.After incubation, the vein in the cord was perfused with plenty ofRinger's buffer and frozen in OCT. The frozen cords were sectioned into10-20 μm slices and mounted on glass slides. The slices were visualizedwith a fluorescence microscope. One-way analysis of variance (ANOVA) wasperformed to determine the statistical significance, p<0.05 wasconsidered to be significant.

Results

PLA-HPG_(ALD) NPs (sticky, also referred to herein as bioadhesivenanoparticles, BNPs) could be reversed to PLA-HPG_(Reversed) (stealth)NPs by NaBH₄ treatment, though one alcohol group is lost with thereduction-reversal cycle since each vicinal diol on HPG is oxidized byNaIO₄ to an aldehyde and each aldehyde is reduced to a single alcohol byNaBH₄. The results are shown in FIG. 8. The blood circulation confirmedthat the PLA-HPG_(ALD) NPs lost almost all their stickiness aftertreatment with NaBH₄. The back and forth tunability also demonstratedthe robustness of the HPG coating on the nanoparticles.

The PLA-HPG NPs (NNPs) without NaIO₄ treatment did not adhere to glassslides and only background signal was detected. However, by transformingthe surface property with NaIO₄, the amount of NPs immobilized on theglass slide increased as a function of duration of NaIO₄ treatment,indicating that the bioadhesive property of the PLA-HPG NPs can be tunedby control of NaIO₄ treatment.

The results are shown in FIGS. 9A, 9B, and 9C. After incubation for 2hours with both NPs and extensive washing, PLA-HPG_(ALD) showedsubstantially higher retention on the luminal side of umbilical veincompared to that of PLA-HPG NPs (P<0.05). The fluorescence intensity wasquantified from the fluorescence images.

The bioadhesive property of PLA-HPG NPs on tissues was evaluated byapplying suspended NPs in vivo to the peritoneal cavity of nude mice.Six BALB/c nude mice were divided into 2 groups, 3 mice per group.IR-780 loaded PLA-HPG_(ALD) NPs (BNPs) and IR-780 loaded PLA-HPG NPs(NNPs) were administrated intraperitoneally into two groups of micerespectively. Each mouse received 100 μL NP suspensions. Thefluorescence was monitored with live imaging (Xenogen) over time. Thequantification of fluorescence retained in intraperitoneal cavity overtime was determined. After IP administration, the majority of PLA-HPGNPs disappeared from the IP cavity within first 24 hours. In contrast,PLA-HPG_(ALD) NPs were retained in the intraperitoneal cavity for atleast 5 days. Even at the end of the 10th day, PLA-HPG_(ALD) NPs werestill detectable at the intraperitoneal cavity. Free dye was also usedas a control but it was all cleared in less than 4 hours.

This result indicates the application of PLA-HPG NPs in local deliverywhere an extended retention at delivery sites is needed. The density ofthe aldehydes on NPs can be controlled thereby providing tunability inthe behavior of the PLA-HPG_(ALD) for local delivery.

These examples show that PLA-HPG_(ALD) NPs will interact with tissuessince the bioadhesive property of PLA-HPG_(ALD) NPs is resulted from theSchiff-base bond between the aldehyde groups on PLA-HPG_(ALD) NPs andthe amine groups in tissue surface.

Example 10. In Vitro Cytotoxicity of Epothilone B-Loaded Nanoparticles

Materials and Methods

Nanoparticle Characterization

The nanoparticles were characterized with TEM with the method describedby Deng et al., Biomaterials 35:6595-6602 (2014). The hydrodynamic sizeof NPs was determined by dynamic laser scattering (DLS).

The amount of EB encapsulated in NPs was determined by HPLC with a C18analytical column (PHENOMENEX®). Acetonitrile/water were used as mobilephase and the wavelength of the UV detector was set at 240 nm.

In Vitro Drug Release

EB loaded NPs suspension (10 mg) in a dialysis tube (10K cut-off) wasdialyzed against 40 ml PBS. At each time point, PBS solution was removedand replaced with 40 ml fresh PBS. The EB released into PBS wasextracted with ethylacetate (EA) twice, 5 ml each. The EA was evaporatedto concentrate the extracts that were then lyophilized. The lyophilizedextracts were dissolved in acetonitrile/water (50:50, volume) mixturefor HPLC analysis.

In Vitro Cytotoxicity of Free EB and EB Loaded Nanoparticles.

UPSC cells (180 μL) were plated in each well of a 96 well plate at adensity of 2,000/well and left in a 37° C. incubator overnight. 20 μl offree EB, EB/PLA-HPG NPs, or EB/PLA-HPG_(ALD) NPs in the medium wereadded to each well. The cells were incubated for 72 h and cell viabilitywas quantified with Cell Titer Blue.

Suppression of Cell Growth by EB/PLA-HPG_(ALD) NPs Attached to LysineCoated Slides.

Six lysine coated slides (25 mm×75 mm) were divided into two groups andeach slide was divided to four quadrants with a pap pen (Abeam). For thefirst group, 100 μl EB/PLA-HPG_(ALD) NPs (1 mg/ml), EB/PLA-HPG NPs (1mg/ml), blank PLA-HPG_(ALD) NPs (1 mg/ml) and PBS; and for the secondgroup, PBS was applied to the quadrants of each slide. After 30 minincubation, each slide was washed extensively with plenty of PBS andplaced to into a 10-cm dish filled with 20 ml medium with a density ofUPSC cells at 2×10⁵/ml. After incubation at 37° C. for 24 hours, themedium was aspirated and the slides were washed with 20 ml PBS for 4times. The cells were stained with Hoechst (for nuclei, blue) andlive/dead stain (green for live cells and red for dead cells) and thenimaged under fluorescence microscope. The number of cells was counted bythe number of nuclei with Image J—particles analysis.

Results

PLA-HPG_(ALD) NPs could be used as a new vehicle of chemotherapy drugsin intraperitoneal delivery since the PLA-HPG_(ALD) NPs have significantextended retention in the intraperitoneal cavity (FIG. 10). Todemonstrate this use of PLA-HPG_(ALD) NPs, epothilone B (EB) was used asthe model drug. EB was encapsulated into the PLA-HPG copolymer to makethe EB/PLA-HPG NPs and then the EB/PLA-HPG NPs were oxidized by NaIO4 toEB/PLA-HPG_(ALD) NPs. The encapsulation efficiency was about 50% and theloading of EB in EB/PLA-HPG NPs and EB/PLA-HPG_(ALD) NPs were 2.5% and1.2% respectively. There was no significant size or morphologydifference between the EB/PLA-HPG NPs and EB/PLA-HPG_(ALD) NPs from bothDLS measurement (Table 2).

TABLE 2 Diameter and polydispersity index (PDI) of EB-containingnanoparticles. Nanoparticles Diameter (nm) PDI EB/PLA-HPG 127 0.225EB/PLA-HPG_(ALD) 127 0.233

The control release curve was measured against PBS. The majority (about80%) of the EB was released after eight hours and there is nosignificant difference between EB/PLA-HPG NPs and EB/PLA-HPG_(ALD) NPs(FIG. 11A). The in vitro cytotoxicity of EB/PLA-HPG_(ALD) NPs againstuterine papillary serous carcinoma (UPSC) cells was also investigated,with EB/PLA-HPG NPs and free EB used as controls. The free EB showed thehighest toxicity (FIG. 11B). The cytotoxicity of EB/PLA-HPG_(ALD) NPswas comparable to that of EB/PLA-HPG NPs. The difference between thefree EB and NP formulations of EB could be due to the retention of theEB in NPs, resulting in a low concentration of EB in the NP formulationscompared to free EB. The enhanced toxicity of EB/PLA-HPG_(ALD) NPs wasdue to the interaction between the PLA-HPG_(ALD) NPs and the cells,resulting in much higher cell uptake of PLA-HPG_(ALD) NPs compared toPLA-HPG NPs.

To confirm that the cytotoxicity is due to the EB, and not the polymers,the effect of blank PLA-HPG_(ALD) NPs and PLA-HPG NPs on UPSC cells wasexamined. Both blank NPs did not show any toxicity up to 1 mg/ml NPs, asshown in FIG. 11C (this concentration of blank NPs was much higher thanthe concentration of the EB loaded NPs used in above studies). Todemonstrate the safety of the PLA-HPG_(ALD) NPs, the cytotoxicity ofblank PLA-HPG_(ALD) NPs (PLA-HPG NPs was used as a control) on humanumbilical vein endothelial cells (HUVEC) and Hela cells was examined.Both blank NPs did not show any toxicity (FIG. 11D). Further, thecytotoxicity of blank PLA-HPG_(ALD) NPs at 1 mg/ml on variety of labcell lines was tested and no cytotoxicity was observed on all these celllines (FIG. 11E).

The cytotoxic efficiency of EB/PLA-HPG_(ALD) NPs was evaluated in vitrousing lysine coated slides with surface-attached EB/PLA-HPG_(ALD) NPsand UPSC cells. Each lysine coated slide was divided into four quadrantswith a hydrophobic pen and EB/PLA-HPG_(ALD) NPs, EB/PLA-HPG NPs, blankPLA-HPG_(ALD) NPs and PBS were applied to each of quadrants. Controlslide with PBS applied to all four quadrants were also prepared. Afterincubation and extensive washes, the slides were exposed to cell culturemedium with UPSC. After 24 hours, the cells on each quadrant were imagedand quantified. There was no significant difference among EB/PLA-HPGNPs, blank PLA-HPG_(ALD) NPs and PBS. However the cell attachment onEB/PLA-HPG_(ALD) NPs coated area was significant suppressed (FIG. 12).The EB released from NPs had no effect to cells in the medium becausethe cell density of PBS control was similar to that of the PBS controlon slides where all four quadrants are applied with PBS. This resultcould be due to the fact that the EB is slowly released fromEB/PLA-HPG_(ALD) NPs and forms a high local drug concentration aroundthe cells closely interacted with EB/PLA-HPG_(ALD) NPs.

These studies confirmed that changing the surface property of NPs tobioadhesiveness could extend the retention of the intraperitoneallyadministrated NPs. Further, the vehicles, the blank PLA-HPG_(ALD) NPs,did not show any cytotoxicity on multiple cell lines for 3 days and upto 1 mg/ml of particles. Moreover, no weight loss or sickness wasobserved when the mice were injected with 5 mgs of blank PLA-HPG_(ALD)NPs intraperitoneally, once a week for 3 weeks. It is believed thenon-toxicity of PLA-HPG_(ALD) NPs may be due to several factors: 1)aldehydes are widely present in foods, fragrances, and metabolites, sohigh tolerance of aldehydes is expected, which is further suggested bythe wide existence of aldehyde dehydrogenase which can detoxifyaldehydes efficiently (Vasiliou et al., Chem Biol Interact 202:2-10(2013)); 2) aldehydes are covalently attached on nanoparticles, althoughthe toxicity of low molecular weight free aldehydes is well known(O'Brien et al., Crit. Rev. Toxicol. 35:609-662 (2005)); and 3) themajority of the aldehyde interaction with proteins or other molecules isthrough reversible Schiff-base bonds so it cannot permanently modify ordamage the biomolecules.

Both EB/PLA-HPG_(ALD) NPs and EB/PLA-HPG NPs have similar size,morphology and controlled release profile. In the regular in vitroassay, both NPs show identical cytotoxicity profile. However, in the invitro assay on lysine coated glass slides, the UPSC cell growth onEB/PLA-HPG_(ALD)NPs coated lysine slides was significantly suppressedwhen compared to the slides treated with EB/PLA-HPG NPs, blankPLA-HPG_(ALD) NPs and control lysine slides. The amount ofEB/PLA-HPG_(ALD) NPs attached on slides was very small and except thearea containing the EB/PLA-HPG_(ALD) NPs, no other area had any effecton cell growth. Adhering to the peritoneum is a prerequisite for thedisseminated cancer cells to establish peritoneal carcinomatosis (“PC”)in the peritoneal cavity (Aoyagi et al., World journal ofgastroenterolog: WJG 20:12493-12500 (2014)). In addition to slowlyreleasing drugs to the whole peritoneal cavity, EB/PLA-HPG_(ALD) NPscould achieve a much higher local concentration of drugs on the surfaceof peritoneum, which could suppress the growth of the cancer cellsadhered to peritoneum and thus eliminate the PC. Further, unlikemicroparticles, which tends to accumulate at lower abdomen, the smallsize of the PLA-HPG_(ALD) NPs allows the diffusion of the NPs to thewhole intraperitoneal cavity.

It is important to emphasize that the unique stickiness of NPs disclosedherein to tissues or tissue mimics is the result of an interactionbetween the stickiness of the aldehyde group and the extremely highdensity of immobilized aldehydes on the NPs. The hyperbranched structureof HPG enables the ratio of aldehyde/PLA to reach 17.

Ex vivo studies confirmed the stickiness of NPs to diverse tissuesurfaces, such as the luminal surface of umbilical vein. No deeppenetration of NPs was observed in these settings, which suggests highlocalization of NPs after topical delivery. Injection of PLA-HPG_(ALD)NPs into the intraperitoneal cavity or subcutaneous tissue resulted inmuch longer retention at these sites. IP administration ofchemotherapeutic drugs such as paclitaxel has become a standardtreatment for certain ovarian and colon cancers (Armstrong, et al., ANZJ Surg, 77:209-213 (2007); Gervais et al., Journal of surgical oncology,108:438-443 (2013)). The standard IP procedure is accomplished through asurgically implanted catheter that allows passage of fluids containingdissolved or suspended chemotherapeutic drug into the abdomen of apatient. In this situation, the retention of the drug depends mainly onthe duration of the infusion. PLA-HPG_(ALD) NPs encapsulated with drugsshould have a great application in this area by significantly extendingthe retention of drugs at the administration site.

Example 11. In Vivo Cytotoxicity of Epothilone B-Loaded Nanoparticles

Materials and Methods

Forty BALB/c Nude mice (5-6 weeks old, Charles River Laboratories) wereintraperitoneally injected with 1×10⁶ UPSC cells. After 1 week, drugtreatments were started with PBS control, blank PLA-HPG_(ALD) controland 3 EB formulations. The injected formulations were as follows: 1) PBS(PH=7.4); 2) blank PLA-HPG_(ALD) in PBS; 3) free EB solution (5 mg/mlstock solution in 30% PEG400/0.5% TWEEN® 80/5% propylene glycol/64.5%water, diluted to needed concentration with PBS before use); 4)EB/PLA-HPG NPs in PBS; 5) EB/PLA-HPG_(ALD) NPs in PBS. Treatments wereadministrated intraperitoneally with a dose of EB (0.5 mg/kg) every weekfor 5 weeks. The body weights of the mice were measured twice a week.Animals were euthanized when the body weight loss exceeded 20%, or whenother signals of sickness, such as breathing problems, failure to eatand drink, lethargy or abnormal posture, were observed.

Results

The Kaplan-Meier survival curve reelecting the efficacy of each of thetreatment formulations is presented in FIG. 13.

In addition to the introduction of a new delivery vehicle, this studydemonstrates that a new PC animal model could be established by IPinoculation of UPSC cells. The highly aggressive characteristics of UPSCinclude early peritoneal or lymphatic spread as well as inbornresistance to chemotherapy. The USPC cell line used in this study is aprimary cell line from a patient. Due to the overexpression of β-tubulinIII, this cell line is resistant to platinum/Taxane but not to EB (Rogueet al., Cancer, 119(14):2582-2592 (2013)). EB, a macrocyclic polyketide,has shown a significantly improved potency for killing UPSC whencompared to paclitaxel. However, the effect of IP administration EB fortreatment of PC is transient because it is a small molecule drug.Therefore, a vehicle with controlled release profile of EB is disclosedherein could achieve better bioavailability of EB for IP delivery.

We claim:
 1. Particles having a diameter between about 25 nm and about250 nm comprising (a) a core comprising one or more hydrophobicbiodegradable polymers; and (b) a shell comprising hyperbranchedpolyglycerol, wherein the hyperbranched polyglycerol is covalently boundto the hydrophobic biodegradable polymer, and and wherein thehyperbranched polyglycerol comprises a plurality of surface hydroxylgroups and vicinal diol groups which decrease uptake of the particles byphagocytic cells following administration to an individual.
 2. Theparticles of claim 1, wherein the biodegradable polymer is an aliphaticpolyester.
 3. The particles of claim 2, wherein the aliphatic polyesteris selected from the group consisting of poly(lactic acid),poly(glycolic acid), and copolymers thereof.
 4. The particles of claim3, wherein the aliphatic polyester is polylactic acid.
 5. The particlesof claim 1, wherein the hyperbranched polyglycerol is covalently boundto the one or more hydrophobic materials.
 6. The particles of claim 1,further comprising one or more agents selected from the group consistingof therapeutic agents, diagnostic agents, prophylactic agents,nutraceutical agents, and combinations thereof.
 7. The particles ofclaim 6, wherein the one or more agents are encapsulated within theparticle, associated with the surface of the particle, or combinationsthereof.
 8. The particles of claim 7, wherein the one or more agents areencapsulated within the particle.
 9. The particles of claim 1, whereinthe particle further comprises one or more targeting agents.
 10. Theparticles of claim 9, wherein the one or more targeting agents arecovalently bound to the shell.
 11. The particles of claim 1, wherein theaverage diameter of the particles is about 100 nm.
 12. A pharmaceuticalcomposition comprising the particles of claim 1 and one or morepharmaceutically acceptable carriers.
 13. The composition of claim 12,wherein the one or more carriers are suitable for parenteraladministration.
 14. The composition of claim 12, wherein the one or morecarriers are suitable for enteral administration.
 15. The composition ofclaim 12, wherein the one or more carriers are suitable for topicaladministration.
 16. A method for delivering one or more agents selectedfrom the group consisting of therapeutic agents, diagnostic agents,prophylactic agents, nutraceuticals agents, and combinations thereof toa patient in need thereof, the method comprising administering aneffective amount of the composition of claim 12, wherein the one or moreagents are encapsulated within the particle, associated with the surfaceof the particle, or a combination thereof.
 17. The particles of claim 1,wherein the surface hydroxyl group density is at least about 1, 2, 3, 4,5, 6, 7, or 8 hydroxyl groups/nm².
 18. The particles of claim 9, whereinthe targeting group density on the shell is at least about 1, 2, 3, 4,5, 6, 7, or 8 targeting groups/nm².